Interactions between metabolism and chromatin in plant models

Journal Pre-proof Interaction between metabolism and chromatin in plant models Christian Lindermayr, Eva Esther Rudolf, Jörg Durner, Martin Groth PII:

S2212-8778(20)30023-5

DOI:

https://doi.org/10.1016/j.molmet.2020.01.015

Reference:

MOLMET 951

To appear in:

Molecular Metabolism

Received Date: 25 October 2019 Revised Date:

10 January 2020

Accepted Date: 24 January 2020

Please cite this article as: Lindermayr C, Rudolf EE, Durner J, Groth M, Interaction between metabolism and chromatin in plant models, Molecular Metabolism, https://doi.org/10.1016/j.molmet.2020.01.015. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier GmbH.

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Interaction between metabolism and chromatin in plant models

2 3

Christian Lindermayr*, Eva Esther Rudolf, Jörg Durner, and Martin Groth*

4 5 6 7

Institute of Biochemical Plant Pathology, Helmholtz Zentrum München – German Research Center for Environmental Health

8 9 10 11 12 13 14 15

*Corresponding Authors:

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Martin Groth Institute of Biochemical Plant Pathology, Helmholtz Zentrum München, Germany Research Center for Environmental Health Ingolstädter Landstrasse 1 85764 München/Neuherberg Tel.: +49 89 3187 49551 Email: [email protected]

Christian Lindermayr Institute of Biochemical Plant Pathology, Helmholtz Zentrum München, Germany Research Center for Environmental Health Ingolstädter Landstrasse 1 85764 München/Neuherberg Tel.: +49 89 3187 2285 Email: [email protected]

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Abstract

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Background: One of the fascinating aspects of epigenetic regulation is that it provides means

35

to rapidly adapt to environmental change. This is particularly relevant in the plant kingdom,

36

where most species are sessile and exposed to increasing habitat fluctuations due to global

37

warming. Although the inheritance of epigenetically controlled traits acquired through

38

environmental impact is a matter of debate, it is well documented that environmental cues

39

lead to epigenetic changes, including chromatin modifications, that affect cell differentiation

40

or are associated with plant acclimation and defense priming. Still, in most case the involved

41

mechanisms are poorly understood. An emerging topic that promises to reveal new insights is

42

the interaction between epigenetics and metabolism.

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Scope of Review: We will review the links between metabolism and chromatin modification,

44

in particular histone acetylation, histone methylation, and DNA methylation, in plants and

45

compare them to examples from the mammalian field, where the relation to human diseases

46

has already generated a larger body of literature. In addition, we will particularly focus on the

47

role of reactive oxygen species (ROS) and nitric oxide (NO) in modulating metabolic

48

pathways and gene activities that are involved in these chromatin modifications. As ROS and

49

NO are hallmarks of stress responses, we predict that they are also pivotal in mediating

50

chromatin dynamics during environmental responses.

51

Major Conclusions: Due to conservation of chromatin modifying mechanisms, mammals and

52

plants share a common dependence on metabolic intermediates that serve as cofactors for

53

chromatin modifications. In addition, plant specific non-CG methylation pathways are

54

particularly sensitive to changes in folate-mediated one-carbon metabolism. Finally, reactive

55

oxygen and nitrogen species may fine-tune epigenetic processes and involve similar signaling

56

mechanisms involved in environmental stress responses in plants as well as in animals.

57

2

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1.

Introduction

59

DNA is packaged inside eukaryotic nuclei by being wrapped around histone proteins.

60

147 base pairs are fitted around each histone octamer consisting of two copies of each core

61

histone H2A, H2B, H3, and H4. The resulting nucleosomes together with associated non-

62

histone proteins and RNA comprise the chromatin. Histones can be modified by

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posttranslational attachment or removal of small chemical groups such as acetyl and methyl.

64

In addition, bases of the DNA molecules are also subject to chemical modification, most

65

prominently cytosine methylation at position 5 of the pyrimidine ring (5-methylcytosine).

66

DNA methylation and posttranslational histone modifications have important functions in

67

gene regulation by influencing the chromatin structure and accessibility of regulatory DNA

68

elements. Transmitted during cell division, chromatin modifications can lead to persistent

69

morphological and physiological changes and are therefore considered as epigenetic marks.

70

The enzymes that catalyze the modifications depend on metabolic intermediates as

71

cosubstrates or cofactors. Although this link between metabolism and epigenetic regulation is

72

evident, the appreciation of the biological implications and underlying mechanisms has just

73

begun. Seminal studies have demonstrated the importance of metabolic regulation in

74

mammalian reprogramming (1, 2). It is now becoming apparent that metabolism not only

75

serve as chemical supplier for chromatin modifications, but that the interaction of central

76

metabolic pathways and epigenetic regulation is decisive for cell fate during development and

77

disease (3).

78

The metabolism of plants is complex and highly adaptive to environmental changes.

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Specialized pathways produce ‘secondary’ metabolites that allow plants to tolerate adverse

80

abiotic conditions, defend themselves against pathogens and herbivores, and communicate

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with their surroundings (4). Secondary metabolic pathways can impose high demands for

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intermediates from primary metabolism. Consequently, primary metabolic pathways have to

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be able to respond dynamically while sustaining central cellular functions (5). Reactive

84

oxygen species, nitric oxide, and glutathione are particularly important for plant

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environmental responses, as they fulfill integral stress signaling functions and regulate

86

cellular redox homeostasis (6). Moreover, photosynthesis implies plant specific pathways and

87

diurnal metabolic patterns, e.g. in connection to starch metabolism (7). As these pathways

88

converge on central intermediates, including S-adenosyl methionine, α-ketoglutarate, and

89

acetyl-CoA, which are known to be require for chromatin modifications, it is likely that plants

90

have evolved epigenetic mechanisms that integrate metabolic dynamics during development

91

and environmental responses. 3

92

Compared to animal models, the territory of plant ‘metaboloepigenetics’ is largely

93

uncharted. So far, the insights are mostly based on forward genetic screens that aimed at the

94

identification of gene silencing mechanisms and revealed many RNA and chromatin-

95

associated factors, but also components of metabolic pathways involved in cofactor regulation

96

(8-12). Approaches that specifically address the connections between metabolism and

97

epigenetics in plants are still sparse. Therefore, this review provides a comparison of current

98

insights in the mammalian and plant field. Due to their prevalence and for the sake of space,

99

we will focus on chromatin acetylation and methylation and the metabolism of the involved

100

cofactors. Finally, we will put the described links between metabolism and chromatin

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dynamics in a biological context to discuss potential causes of environmentally induced

102

epigenetic changes and how these changes might contribute to stress resistance in plants.

103 104

2.

Histone acetylation and acetyl-CoA metabolism

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2.1.

Overview of histone acetylation and deacetlytion in plants

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Posttranslational modifications (PTMs) of histone proteins include acetylation,

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methylation, sumoylation, ubiquitinylation, phosphorylation, glycosylation, and carbonylation

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of different histone residues. The addition and removal of these modifications by chromatin-

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modifying complexes makes the chromatin structure very dynamic and allows fine-tuning of

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the accessibility of the DNA to transcription factors. Because of the important function of

111

histone acetylation on chromatin structure, this modification has been intensively investigated

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already for several decades. Acetylation of histones typically occurs at the N-terminal tails,

113

which are rich in lysine residues, and neutralizes the positive charges of the histone tails

114

resulting in a decrease of their affinity for negatively charged DNA. Thus, acetylation of

115

histones is associated with a loosened chromatin structure and often occurs in the promoter

116

and 5’-end of genes enabling transcription factors binding to DNA (13). Moreover, acetylated

117

histone lysine residues can be recognized by transcriptional co-regulators and chromatin

118

remodeling factors containing a ‘bromodomain’ (14).

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The level of histone acetylation is determined by the activity of both histone acetyl

120

transferases (HATs) and histone deacetylases (HDACs). HATs use acetyl-CoA as cofactor to

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transfer acetyl groups on histone-lysines. They can be classified into four distinct families,

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harboring different substrate specificities (15-17). For instance, HAG1 from the GCN5-like

123

family almost exclusively acetylates H3K14, whereas HAG2 from the same family shows

124

specificity for H4K12. HAM1 and HAM2 belonging to the Myst-like family of HATs both

125

acetylate H4K5, whereas HAC1, 5 and 12 from the CBP-like family have multiple target 4

126

lysines on H3 and H4. The fourth group called TAFII250-like has not been characterized in

127

plants. Some HATs contain bromodomains, enabling them to bind acetylated histones,

128

indicating that acetylation of histones by one HAT may help to recruit other HATs on the

129

same nucleosome (15).

130

The HDACs in Arabidopsis thaliana (herein after just Arabidopsis) are encoded by 18

131

genes and can be grouped into three types, including silent information regulator 2 (SIR2),

132

reduced potassium dependency 3 (RDP3), and the plant-specific histone deacetylase 2 (HD2)

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(16, 17). The structurally diverse family of sirtuins (2 members in Arabidopsis) contains

134

homologs to yeast SIR2. These enzymes catalyze protein deacetylation using NAD+ as

135

cofactor. The RPD3 family consists of 12 members and is characterized by a highly

136

conserved HDAC domain with a catalytic zinc ion (15-17). The HD2-like family (HD2A,

137

HD2B, HD2C, and HD2D) is plant specific, since no homologs have been identified in other

138

organisms so far (17). Although HD2-ko plants have altered histone acetylation levels, their

139

HDAC activity is still questioned. Maybe they are important interaction partners of RPD3-

140

like HDACs required for their activity.

141

Analysis of mutants affected in either HAT or HDAC revealed that histone acetylation

142

plays key roles in a number of physiological processes, including cell cycle, flowering time,

143

response to environmental conditions such as light or pathogen attack, root and shoot

144

development, hormone signaling (16). These functions of HATs and HDACs are not only

145

associated with chromatin modification, because HATs and HDACs have been found to be

146

regulating also the acetylation of numerous non-histone proteins.

147 148

2.2.

Acetly-CoA metabolism affects histone acetylation in plants

149

Acetyl-CoA is a central metabolite involved in numerous anabolic and catabolic

150

pathways and is a substrate for protein acetylation. In mammalian cells, the nuclear and

151

cytosolic acetyl-CoA is supplied by at least three pathways: citrate cleavage, pyruvate

152

dehydration and acetylcarnitine conversion (18-20). In cytosol and nucleus, adenosine

153

triphosphate (ATP)-citrate lyase (ACL) cleaves citrate exported from mitochondria to

154

regenerate acetyl-CoA that can be used for other biosynthetic processes, such as fatty acid

155

synthesis and histone acetylation (21). Moreover, a pyruvate dehydrogenase complex can be

156

translocated from mitochondria to nuclei to generate acetyl-CoA and mediate histone

157

acetylation in mammalian cells in certain conditions (20, 22).

158

In plant cells, plastids, mitochondria, peroxisomes, and the cytosol produce acetyl-

159

CoA (23). In Arabidopsis, it has been shown that cytosolic acetyl-CoA is mainly produced by 5

160

the ATP-citrate lyase and that reduction of ACL activity leads to complex bonsai phenotypes

161

(23, 24). Cytosolic acetyl-CoA can either be converted to malonyl-CoA by acetyl-CoA

162

carboxylase (ACC) to synthesize very-long-chain fatty acids, flavonoids, and malonyl

163

derivatives, or condensed to acetoacetyl-CoA for synthesizing precursors of isoprenoids (25,

164

26). Two different isoforms of ACC have been described until now. The heteromeric ACC is

165

composed of four subunits and is located in the plastids where it is responsible for de novo

166

fatty acids synthesis. The homomeric ACC is composed of a single large polypeptide

167

containing ACC1 and ACC2 and is located in the cytosol. It is required for elongating the

168

plastid-produced fatty acids. Blocking the function of cytosolic ACC1 leads to elevated levels

169

of acetyl-CoA and, consequently, to global histone hyperacetylation, predominantly at lysine

170

27 of histone H3 (H3K27), as shown in Arabidopsis (27). The increase of H3K27ac in acc1

171

mutants is dependent on ACL and the histone acetyltransferase GCN5. Consequences of the

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increased acetyl-CoA level and H3K27ac are changes in gene transcription and metabolite

173

levels. Gene ontology analysis indicates that H3K27 hyperacetylated genes are enriched in

174

primary metabolic processes including amino acid biosynthesis. Consistently, expression of

175

22 amino acid biosynthesis genes is at least twofold increase in acc1 (27). Furthermore, two

176

of the tricarboxylic acid (TCA) cycle intermediates (isocitrate and α-ketoglutarate) are

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significantly over accumulated in acc1. Isocitrate is metabolized to α-ketoglutarate by

178

isocitrate dehydrogenase. Interestingly, α-ketoglutarate is an important metabolic intermediate

179

that acts as a cofactor for several chromatin-modifying enzymes, including histone

180

demethylases (28).

181

Another study has revealed a link between peroxisomal acyl-CoA oxidase 4 (ACX4),

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an enzyme in the fatty acid β-oxidation pathway, histone acetylation and DNA methylation-

183

mediated gene silencing (29). Screening for suppressor of pro35S:NPTII silencing the authors

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identified an acx4 mutant with reduced overall levels of H3Ac and H4Ac and increases DNA

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methylation at some endogenous genomic loci, which results in enhanced transcriptional

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silencing of reporter and some endogenous genes (29). Thus, ACX4 activity of the fatty acid

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β-oxidation in peroxisomes is closely linked to nuclear epigenetic modifications, which may

188

affect diverse cellular processes in plants. In sum, fluctuation of acetyl-CoA levels in the

189

cytosol or organelles affect histone acetylation in eukaryotic cells (24, 27, 30).

190 191

3.

Chromatin methylation and one-carbon metabolism

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3.1.

DNA and histone methylation pathways in plants

193

DNA and histone methylation, in addition to histone acetylation, are among the most 6

194

studied and most conserved chromatin modifications. Although DNA methylation has been

195

lost in some eukaryotes, its common role is the silencing of transposable elements. Moreover,

196

DNA methylation is associated with additional functions that involve long-term gene

197

inactivation, such as imprinting and X-inactivation. Loss of DNA methylation is embryo

198

lethal in mammals and often leads to severe developmental defects in plants, but is tolerated

199

in Arabidopsis, which makes it a very useful model for genetic dissection of the involved

200

pathways. In contrast to mammals, where DNA methylation is almost exclusively found at

201

CG sites, most plants contain also CHG and CHH (H = C, A or T) methylation (31). In

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Arabidopsis, DNA methylation in all sequence contexts is established by the RNA-directed

203

DNA methylation (RdDM) pathway, which includes the plant-specific Pol II-related RNA

204

polymerases Pol IV and V, and components of RNA interference (31). Once established,

205

symmetric CG methylation (i.e. CG sites that are methylated on both strands) is propagated

206

during replication by recruitment of the maintenance DNA methyltransferase MET1 (a

207

homolog of mammalian DNMT1) to hemimethylated DNA at the replication fork (31). This

208

mechanism is conserved between mammals and plants. CHH methylation is either maintained

209

by RdDM, mostly acting on the euchromatic chromosome arms, or by the plant-specific DNA

210

methyltransferase CMT2, whereas CHG methylation is maintained by its paralog CMT3 (31).

211

Cross-talk between and self-reinforcing loops within the DNA methylation pathways ensure

212

maintenance of non-CG methylation patterns. The involved feedback mechanism is illustrated

213

by the interplay of non-CG methylation and dimethylation of lysine 9 on histone H3

214

(H3K9me2), which is catalyzed by the SET domain-containing histone methyltransferases

215

(HMTs) KYP/SUVH4, SUVH5 and SUVH6: CMT2 and CMT3 bind nucleosomes containing

216

H3K9me2 deposited by the HMTs, which in turn bind to cytosines methylated by CMT2 and

217

CMT3 (32). This mechanism leads to the genome-wide association of non-CG and H3K9

218

methylation (33). Moreover, mammalian genomes undergo global erasure and re-

219

establishment of most DNA methylation marks in pre-implantation development and during

220

gametogenesis. In contrast, global resetting of DNA methylation in Arabidopsis does not

221

seem to occur in generative nuclei of the gametes or during embryogenesis (34). Accordingly,

222

DNA methylation patterns are generally stable and transgenerational inheritance of epialleles

223

is common in Arabidopsis and other plants (34).

224

Histone methylation marks are commonly found on arginine and lysine residues of the

225

N-terminal tails of histones H3 and H4 (for the sake of space, we will hereinafter only

226

consider lysine methylation). Lysine methylation in general depends on methyltransferases

227

with a catalytic SET domain and surrounding conserved sequences that together specify the 7

228

target and number of methyl groups added (up to 3) (35). Depending on the position of the

229

lysine residue and degree of methylation, these marks are associated with different

230

transcriptional states (36). For example, highly conserved H3K4 methylation is enriched in

231

promoters of active genes, whereas H3K27me3 marks inactive genes that are controlled by

232

the Polycomb Group proteins and often have important developmental functions in animals as

233

well as plants (37, 38).

234

DNA and histone methylation are reversible and the active reversal plays important

235

roles during development and environmental responses. As mentioned above, animal and

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plant genomes display fundamental differences in their DNA methylation dynamics, which

237

are also reflected in their DNA demethylation mechanisms. Animals rely on ten-eleven

238

translocation

239

hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (39-41). Once oxidized,

240

replication leads to ‘passive’ replacement of 5-methylcytosine by cytosine. Alternatively, 5-

241

formylcytosine, and 5-carboxylcytosine are actively excised and replaced by cytosine through

242

thymine DNA glycosylase and base excision repair (42). Plants, in contrast, are lacking genes

243

that encode TET proteins (39). Instead, active DNA demethylation is achieved through the

244

excision of 5-methylcytosine by a family of DNA glycosylases, including DME, DML2 and

245

3, and ROS1, followed by cytosine replacement through the base excision repair pathway

246

(43). DME activity in the central cell ensures genomic imprinting in the endosperm and is

247

essential for seed development (44). The main function of ROS1 appears to be the

248

confinement of DNA methylation to its target region in order to avoid DNA methylation

249

spreading and silencing of adjacent genes (45).

(TET)

proteins

that

can

stepwise

oxidize

5-methylcytosine

to

5-

250

Histone demethylation involves the hydroxylation of methyl groups by histone

251

demethylases belonging either to the class of flavin adenine dinucleotide (FAD)-dependent

252

amine oxidases or the Fe(II) and α-ketoglutarate-dependent hydroxylases (46). Members of

253

the first class include LSD1/KDM1 homologs, which play critical roles in cell differentiation

254

and use mono- and dimethylated H3K4 and H3K9 as substrates. Members of the second class

255

are characterized by the Jumonji (Jmj) domain that binds the cofactors Fe(II) and α-

256

ketoglutarate. They form several families that are distinguished by domain architecture and

257

substrate specificity (46). In Arabidopsis, about half of the 21 Jmj-coding genes and all four

258

LSD1 homologs have been characterized so far. The described functions, frequently fulfilled

259

by partially redundant paralogs, range from DNA methylation and transposon silencing to

260

negative regulation of flowering and circadian clock genes (47-50).

261 8

262

3.2.

Changes in the methionine cycle affect transcriptional gene silencing in plants

263

S-adenosylmethionine (SAM) serves as methyl donor for most enzymatic methylation

264

reactions, including cytosine methylation and lysine methylation by DNA and histone

265

methyltransferases, respectively (Figure 1). Accordingly, there is wide evidence, ranging from

266

Drosophila, Caenorhabditis elegans, and mammalian models to rice and Arabidopsis, that

267

deficiencies in SAM production lead to reduced chromatin methylation (11, 51-53). SAM is

268

the central constituent of the methionine cycle and produced by S-adenosylmethionine

269

synthetase (SAMS/MAT) from methionine (Met) and adenosine triphosphate (ATP) as

270

substrates. Among the four SAMS isoforms in Arabidopsis, AtSAMS4 seems to be

271

particularly required for DNA and histone methylation (54). As reported by the authors,

272

mutations in AtSAMS4 led to decreased levels of SAM, globally reduced non-CG

273

methylation and H3K9me2, and, accordingly, activation of many TEs. Other SAMS isoforms,

274

in contrast, were dispensable for TE silencing, which might be due to functional redundancy

275

or reduced and tissue-specific expression (54).

276

Upon methyl transfer, SAM is converted to S-adenosylhomocysteine (SAH), which

277

per se functions as a competitive inhibitor of SAM-dependent reactions, due to higher

278

affinities of most methyltrasferases towards SAH than SAM (55). Accordingly, tissue or

279

plasma SAM/SAH ratios (also known as methylation index) are often correlated with

280

methylation levels, particularly DNA methylation, and can therefore serve as indicators of the

281

methylation status (56, 57). SAH is reversibly converted to adenosine (Ado) and

282

homocysteine (Hcy) by the enzyme SAH hydrolase (SAHH) (58). As this constitutes the only

283

way to remove SAH, blocking SAHH activity leads to increased SAH levels, which is often

284

accompanied by feedback accumulation of SAM and Met, but overall leads to decreased

285

SAM/SAH ratios. In plants, DNA methylation and methylation of histone H3 lysine 9

286

(H3K9me) seem to be particularly sensitive to impaired SAHH activity, as Arabidopsis sahh1

287

mutants have been identified multiple times in different screens for transcriptional gene

288

silencing factors (8, 59-61). Moreover, expression of antisense RNA of SAHH in tobacco

289

plants resulted in a loss of DNA methylation in repetitive elements (62). Other studies

290

employed a selective reversible inhibitor of SAHH, dihydroxypropyladenine, in tobacco

291

callus and TBY-2 cells, leading to accumulation of SAH

292

hypomethylation (63, 64), as well as pleiotropic developmental defects, such as decreased

293

apical dominance, altered leaf and flower symmetry, and reduced fertility in in treated

294

tobacco plants (65). Arabidopsis contains two SAHH isoforms encoded by SAHH1 and

295

SAHH2. SAHH1 appears to be the main isoform as only SAHH1 is an essential gene and 9

and concomitant DNA

296

generally expressed at higher levels than SAHH2 (66). Characteristically, sahh1 knock-down

297

or hypomorphic mutants display derepression of many transposable elements (TEs) due to

298

global reduction in H3K9me2 and DNA methylation, non-CG methylation being mostly

299

affected. This is probably due to a simultaneous inhibition of the respective HMTs

300

(KYP/SUV4, SUVH5, and SUVH6) and DNMTs (CMT2 and 3) that is caused by SAH

301

accumulation.

302

methyltransferases constitutes an ‘Achilles heel’ of the feedback mechanisms, which

303

normally ensures stability of non-CG methylation patterns (10). Consequently, manipulation

304

of SAH levels constitutes an efficient way to alter non-CG methylation.

In

other

words,

shared

SAM

/SAH-sensitivity

of

the

involved

305

The reaction catalyzed by SAHH thermodynamically favors SAH production (58). In

306

order to avoid feedback accumulation of SAH, Hcy and Ado have to be rapidly removed. Ado

307

is removed by conversion to adenosine monophosphate (AMP), which is catalyzed by ATP-

308

dependent Ado kinase (ADK) and constitutes the main salvage pathway in plants.

309

Alternatively, Ado can be converted to inosine by Ado deaminase, but this path is not present

310

in plants (67, 68). Reduced ADK activity in transgenic Arabidopsis lines was associated with

311

dwarf phenotypes, SAH accumulation and reduced pectin methylation of seed mucilage (67).

312

In animals, Hyc is removed either by conversion to cysteine (Cys) via the reverse

313

transsulfurylation pathway, or by being recycled to Met, either in a 5-methyltetrahydrofolate

314

(5-CH3-THF)-dependent reaction that is catalyzed by methionine synthase (MS), or by folate-

315

independent betaine-homocysteine S-methyltransferase homologs (69). Due to the lack of

316

reverse transsulfurylation, Hcy levels in plants are controlled by 5-CH3-THF-dependent MS

317

activity as part of the methionine cycle and by homocysteine S-methyltransferase as part of

318

the S-methylmethionine cycle (70).

319

MS from animals require cobalamin (vitamin B12) as cofactor for activation. As

320

cobalamin is prone to non-catalytic oxidation, sustained MS activity requires regeneration of

321

active cobalamin by MS reductase (encoded by Mtrr). Several mammalian studies have

322

pointed towards an important role of cobalamin-dependent MS activity for epigenetic

323

regulation (71-73). A hypomorphic Mtrr mutation in mice has been associated with

324

congenital malformations in wild-type grand-progeny that were associated with altered DNA

325

methylation and persisted for two additional generations (74). In another study, genome-wide

326

association mapping of sequence variation and CG methylation patterns in 90 mouse strains

327

revealed a quantitative trait locus hotspot near Mtrr. Correlation with Mtrr expression further

328

indicated that sequence variation at Mtrr affects CG methylation patterns (75). 10

329

In contrast, MS enzymes described in higher plants and fungi are cobalamin-

330

independent. Nevertheless, the importance of proper Hcy recycling for the maintenance of

331

DNA methylation was recently confirmed by a genetic screen for Arabidopsis silencing

332

mutants the led to the identification of a MS hypomorph (12). Accordingly, the identified ms1

333

mutant showed decreased Met and increased Hcy content, along with decreased SAM/SAH

334

and epigenetic defects that were reminiscent of other mutants impaired in the methionine

335

cycle. In agreement to this and other studies, MS1 seems to play a predominant role in Hcy

336

recycling, as loss-of-function is lethal and not compensated by the other two paralogs in

337

Arabidopsis, which were not required for DNA methylation or viability in general (12, 76).

338 339

3.3.

The methionine cycle, and hence transcriptional gene silencing, depend on folate

340

metabolism

341

Folate metabolism provides one-carbon (C1) units for the synthesis of nucleotides,

342

initiator tRNA, pantothenate, as well as for the recycling of Hcy to Met (70). The C1 units are

343

carried by different intermediates of tetrahydrofolate (THF), ranging from the reduced 5-CH3-

344

THF to the fully oxidized 10-formyltetrahydrofolate (10-CHO-THF). The corresponding C1-

345

dependent pathways are compartmentalized in the cyto- and nucleoplasm, mitochondria, and

346

plastids. The subcellular folate pools fulfill specific metabolic functions that are reflected in

347

the THF composition (77, 78). For example, 5-CH3-THF is predominant in the cytoplasm,

348

where it acts as methyl donor for Hcy methylation as part of the methionine cycle (79-81).

349

Plant vacuoles also contain 5-CH3-THF, which probably serves solely as storage of 5-CH3-

350

THF from the cytoplasm (81). 5-CH3-THF is produced from 5,10-CH2-THF by

351

methylenetetrahyrofolate reductase (MTHFR). In contrast to mammals, where this reaction is

352

irreversible, there is evidence that MTHFR activity is reversible in plants (82). Cytoplasmic

353

5,10-CH2-THF (the precursor of 5-CH3-THF) can be either produced by MTHFD1 or by

354

serine hydroxymethyltransferase (SHMT), which reversibly converts THF and serine (Ser) to

355

5,10-CH2-THF and glycine (Gly) (70).

356

Since the methionine cycle depends on C1 supply from 5-CH3-THF, dietary and

357

genetic folate deficiencies in humans and mice are frequently associated with elevated plasma

358

Hcy levels (83) and can cause DNA hypomethylation (84). In Arabidopsis, inhibition of folate

359

biosynthesis by sulphonamide sulfamethazine (SMZ) as well as a loss-of-function mutation in

360

FPGS1, which encodes the plastidic folylpolyglutamate synthetase required for cellular folate

361

homeostasis, leads to DNA and H3K9 hypomethylation and TE activation (9, 85, 86). Rescue

362

by supplementation of growth media with folinic acid (5-CHO-THF), which is readily taken 11

363

up and converted to 5-CH3-THF, suggest that the epigenetic defects caused by SMZ and loss

364

of FPGS1 function are linked to folate limitation (9, 85, 86). The hypomorphic mthfd1-1

365

mutant, which also has been identified by forward genetic screening for silencing defects,

366

shows DNA and H3K9me2 methylation losses, associated with Hcy and SAH accumulation.

367

Although this is reminiscent of other mutants impaired in folate-dependent Met cycling, total

368

leave tissue folate content, and in particular levels of 5-CH3-THF were not altered in mthfd1-

369

1. Moreover, supplementation with folinic acid did not rescue the mthfd1-1 phenotype, which

370

raised the open question, whether folate limitation causes Hcy accumulation in mthfd1-1, or

371

whether MTHFD1 might affect Hcy recycling by another mechanism (10).

372

The complexity of folate metabolism with its multiple interdependent subcellular

373

pathways is reflected by the SHMT gene family in Arabidopsis, which contains seven

374

members that have partially redundant compartment-specific functions. Among them,

375

SHMT6 and SHMT7 are predicted to localize to the nucleus. Intriguingly, shmt7/msa1

376

Arabidopsis mutants, which have been identified during a genetic screen for leaf elemental

377

composition and accumulated sulfur due to constitutive upregulation of sulfate assimilatory

378

pathways, showed slightly reduced DNA methylation levels in roots that were associated with

379

lower root tissue SAM levels (87). Although SHMT7 did not show in vitro activity, the

380

authors suggested the SHMT7 might be involved in nuclear SAM supply and that sulfur

381

homeostasis is regulated through a DNA methylation- and SAM-dependent mechanisms (87).

382 383

4.

Redox-dependent chromatin modulation

384

4.1.

Principles of redox-signaling

385

Redox reactions are evolutionarily conserved signaling principles, occurring in

386

prokaryotes and eukaryotes. The most important redox molecules are reactive oxygen species

387

(ROS) and nitric oxide (NO). ROS and NO are produced during normal energy metabolism

388

(e. g. respiration, photorespiration, and photosynthesis) and stress responses in plant cells.

389

Cellular redox processes are extremely sensitive to environmental conditions. The nuclear

390

compartment that orchestrates genetic programs of cell life is on one side particularly

391

sensitive to the deleterious effects of oxidation and on the other side contains fine-tuned

392

redox-regulated signaling pathways. Experiments using isolated tobacco nuclei demonstrated

393

that plant nuclei are an active source of ROS production, in particular of H2O2 (88) and

394

increasing evidence suggests an important role of redox signaling in the regulation of many

395

nuclear proteins like kinases, transcription factors and chromatin-modulators (89, 90).

396

Modification of the cysteine redox state is the predominant redox-signaling mechanism. 12

397

Solvent exposed thiols are prone to oxidation especially in a basic environment, which is

398

promoting thiol deprotonation and formation of a thiolate residue (R-S-). This nucleophilic

399

residue is very sensitive to oxidation.

400

In the presence of ROS, such as H2O2 or superoxide, thiol groups are oxidized to

401

sulfenic (R-SOH), sulfinic (R-SO2H) and sulfonic (R-SO2H) acids. Moreover, thiols can also

402

be oxidized by oxidized glutathione (GSSG) resulting in S-glutathionylation (R-S-SG) (91,

403

92). Two closely located cysteine residues can also get oxidized to form a disulfide bridge (S-

404

S). All these modifications can alter the structure and/or the activity of proteins in all the

405

cellular compartments, including chromatin modifiers in the nucleus.

406

NO is a ubiquitous signaling molecule with pleiotropic functions throughout the

407

lifespan of plants. It is involved in several physiological processes, including growth and

408

development, biotic and abiotic stress response as well as in iron homeostasis (93-103). The

409

multifunctional role of NO is based on its chemical properties, cellular environment, and

410

compartmentation (104, 105). NO has been described as cytoprotective as well as cytotoxic

411

molecule depending on its local concentration, which is affected by its rate of synthesis,

412

displacement, and removal (105, 106). The radical nature of NO promotes its interaction with

413

different macro-molecules to transduce its bioactivity. Endogenously synthesized or

414

exogenously applied NO is able to react with other radicals, including ROS resulting in the

415

formation of reactive nitrogen species (RNS) or with metals forming metal-nitrosyl

416

complexes such as dinitrosyl-iron complexes (DNIC; formed by the interaction between NO,

417

iron, and thiol-containing ligands such as glutathione (GSH), cysteine, or protein thiols) (107,

418

108). NO exerts its biological function through post-translational modifications (PTMs),

419

including tyrosine nitration, metal nitrosylation, and S-nitrosation. Those NO-mediated PTMs

420

have profound effects on the function of target proteins by regulating their activities,

421

subcellular localization, structure, or interaction with biomolecules and consequently, induce

422

diverse physiological responses and/or signaling processes, including alteration of gene

423

expression metabolic changes, and phytohormone signaling (109-116). The most intensively

424

studied signal transduction mechanism of NO is protein S-nitrosation. Proteome-wide studies

425

identified putative S-nitrosated proteins involved in primary metabolism, defense response

426

and regulation of transcription (117-120). Moreover, in more detailed studies it has been

427

demonstrated that NO regulates transcription directly by modifying transcription factors, such

428

as TGA1 (121), MYB30 (122), SRG1 (123). However, several lines of evidence indicate that

429

NO regulates gene expression also via modification of chromatin accessibility (114, 124-127).

430

Consequently, the redox status of plant cells has the potential to control chromatin 13

431

modifications and epigenetic reprogramming of gene expression (128-133). In the following

432

paragraphs we discuss the regulatory function of redox molecules on covalent modifications

433

of core histones, DNA-methylation, and metaboloepigenetic effects.

434 435

4.2.

Redox-regulation of histone acetylation

436

Redox regulation of epigenetic processes has mostly been addressed in mammals

437

(124-129). Multiple studies have provided evidence that histone deacetylation activities are

438

sensitive to redox modification resulting in altered chromatin conformation and

439

transcriptional changes (125, 134, 135). Different enzymes responsible for regulating H3K9ac

440

and H3K14ac seem to be targets for redox regulation. For example, a ROS/thioredoxin

441

(TRX)-dependent redox switch of key Cys residues regulatesd nuclear trafficking of the class

442

II HDAC4 (135) and S-nitrosation of HDAC2 at conserved cysteine in neurons affects histone

443

modification and gene expression (125). In plants, however, exploration of this research topic

444

just started a few years ago (28, 133, 136, 137). Since several histone acetylation mechanisms

445

are largely conserved in eukaryotes, it is likely that distinct plant HDACs are also redox-

446

regulated. Within the 18 identified HDACs in plants (17), members of the class I RPD3-like

447

HDAC (HDAC6, HDAC9, HDAC19) might be sensitive to oxidation (114, 133, 138). Human

448

and plant HDACs share a domain, which contains six highly conserved cysteine residues.

449

Interestingly, the cysteine residues, which are targeted by NO in human HDAC2 (Cys262 and

450

Cys274) are located within this region and structural modeling of the plant HDAC domain

451

based on the available crystal structure of human HDAC2 revealed a strikingly similar 3D-

452

fold of these proteins (133). The two conserved potential NO-sensitive cysteine residues of

453

plant HDACs are located close to the substrate binding site at the same positions as in human

454

HDAC2. This makes plant HDAC6, HDCA9 and HDAC19 promising candidates for NO-

455

affected/regulated nuclear HDAC isoform(s).

456

Chemical treatment with an NO-donor, S-nitrosoglutathione (GSNO), and subsequent

457

demonstration of specific changes in H3K9/14 acetylation pattern in Arabidopsis seedlings

458

by ChIP-seq has been done to further investigate the effect on histone acetylation (114). Most

459

of the observed alterations were found within genes involved in the response to biotic and

460

abiotic stresses. The majority of NO-regulated H3K9/14ac sites were similarly affected by

461

trichostatin A treatment (a strong inhibitor of RPD3/HDA1-like HDACs), indicating that

462

these changes might be induced by NO-dependent inhibition of HDAC activity. Supporting

463

this, a slight but significant increase in total H3ac was observed upon treatment of

464

Arabidopsis suspension cells with GSNO. Moreover, salicylic acid induced a rapid and strong 14

465

endogenous NO production in plant cells, which correlated with a decrease in HDAC activity,

466

and scavenging of NO or treatment with a reducing agent rescued HDAC activity, indicating

467

that the inhibition was mediated by S-nitrosation (114). In conclusion, NO-dependent

468

regulation of plant HDACs can be considered as a key mechanism in regulation of histone

469

acetylation and gene expression. NO-dependent regulation of plant HDACs might play a role

470

during the plant´s stress response to enhance transcription of stress-related genes by inducing

471

histone acetylation at the corresponding loci.

472 473

4.3.

Redox-dependent modulation of histone and DNA methylation

474

In mammals, NO has been shown to directly and indirectly affecting DNA and histone

475

methylation (reviewed in (128-132)). Indeed, NO induces transcriptional changes of DNA

476

and histone methyltransferases (HMTs) and demethylases. For example, the expression levels

477

of H3K9me HMTs are differentially controlled by NO in human cells. Upon treatment with

478

NO, the expression levels of SETDB2 and SUV39H2 (both tri-methylate H3K9) increased,

479

while the levels of G9a (di-methylates H3K9) decreased (132, 139). Application of RRX-001,

480

a compound generating reactive nitrogen and oxygen species, decreased the expression of

481

DNA methyltransferases (DNMTs) in mammalian cells and diminished global DNA

482

methylation levels (140). Conversely, exposure of mammalian cells to NO-donor SNAP did

483

not change the expression of DNMTs (141).

484

Furthermore, NO-induced alteration of enzymatic activities of chromatin-modifying

485

enzymes was observed. For instance, the activity of DNMTs increased when NO was applied

486

directly to a nuclear protein extract (141). Similarly, endogenously produced NO upon

487

Helicobacter pylori treatment was associated with increased DNMT activity and an increase

488

in DNA methylation (142). Multiple studies have indicated that HMTs are also prone to redox

489

modification (143-145). Moreover, it is now well established that NO inhibits mononuclear

490

non-heme iron dioxygenases such as JHDM (histone demethylase) and TET (DNA-

491

demethylase) enzymes by the formation of a nitrosyl-iron complex with their catalytic non-

492

heme iron (139, 146). For instance, the NO-mediated inhibition of recombinant human lysine

493

demethylase 3A (KDM3A) by forming a nitrosyl-iron complex in the catalytic pocket

494

prevents the binding of the co-substrate O2 required for demethylase activity (139).

495

Accordantly, exposing cells to NO resulted in a significant increase in H3K9me2, the

496

preferred substrate for KDM3A (139). Similarly, the NO donor DETA-NO caused global

497

increases in the levels of H3K9me2, H3K9me3, and H3K36me3 to the same extent as the

498

dioxygenase-inhibitor dimethyloxallyl glycine (α-KG analog) in macrophages. Additionally, 15

499

NO affect the activities of JmjC domain-containing histone demethylase (JHDMs) by

500

sequestration of chelatable iron, which is considered the major source of iron for JHMDs, via

501

the formation of dinitrosyl-iron complexes (139).

502

NO can also lead to enzymatic degradation, as shown for the H3K9 tri-methylating

503

HMT SUV39H1. Neurotrophins-induced NO production resulted in S-nitrosation of GAPDH

504

enabling its nuclear translocation in complex with SEVEN IN ABSENTIA HOMOLOG 1

505

(SIAH1) protein, a known ubiquitin E3 ligase. The GAPDH-SIAH1 complex associated with

506

SUVH39H1 and mediated its degradation, which in turn resulted in decreased H3K9me3

507

levels (147). Taken together, redox events have emerged as important mechanisms to regulate

508

chromatin modifiers on different levels (transcription, activity and degradation of chromatin

509

modifiers) resulting in altered histone methylation pattern and DNA methylation in

510

mammalians.

511

In plants, different studies have analyzed transcriptome changes upon exogenous NO

512

application or in plants with impaired NO homeostasis. Interestingly, many of the

513

differentially expressed genes had annotations linked to DNA or histone methylation

514

(summarized in Table). For instance, genes involved in de novo and maintenance DNA

515

methylation as well as in active demethylation were differentially expressed upon treatment of

516

Arabidopsis leaves with the NO-donor CysNO (148). Intriguingly, de novo methylation and

517

maintenance pathways showed opposite effects, as various components and positive

518

regulators

519

DEMETHYLASE 1-LIKE 1 (LDL1), were down-regulated, whereas CMT2 and AGENET

520

DOMAIN-CONTAINING

521

heterochromatin (149), were upregulated. Additionally, CysNO treatment resulted in down-

522

regulation of ROS1, the major DNA demethylase in Arabidopsis (148). It might therefore be

523

that NO exerts different regulatory effects during establishment and maintenance of DNA

524

methylation or on euchromatic vs. pericentromeric heterochromatin, as the former is targeted

525

by RdDM, whereas the latter is methylated by CMT2.

of

the

RdDM

P1,

pathway,

which

including

links

SUVH9

H3K9me2

and

to

LYSINE-SPECIFIC

DNA

methylation

in

526

Further, CysNO altered the expression of genes encoding JHMDs (148). In regard that

527

JHMDs require iron for their activity, it is noteworthy that genes encoding NICOTINAMINE

528

SYNTHASE (converts SAM into nicotianamine) isoforms are differentially regulated upon

529

NO donor treatment (148, 150) and in mutants with impaired NO homeostasis (150-154).

530

Nicotianamines are high-affinity metal chelator molecules and play a key role in Fe

531

homeostasis and transport in plants. Transcriptomic analysis of NO-deficient noa1-2,

532

nia1nia2, and nia1nia2noa1-2 mutants also revealed that genes encoding for enzymes 16

533

involved in epigenetic methylation processes are differentially expressed (155). For instance,

534

CMT2, responsible for CHH methylation at pericentromeric heterochromatin, were down-

535

regulated in each mutant (155). Additionally, MET1 maintaining CG methylation was

536

upregulated in noa1-2, but downregulated in nia1nia2. Further, genes encoding for enzymes

537

involved in active DNA demethylation such as ROS1 and DML2 were differently expressed

538

in these NO-deficient mutants. Further, numerous genes encoding essential components of the

539

CIA pathway were upregulated in nia1nia2noa1-2. The CIA pathway is required for

540

maturation of Fe-S cluster proteins such as ROS1 (156). Interestingly, expression of several

541

protein arginine methyltransferases (PRMTs) were up-regulated in NO-deficient plants. For

542

instance, PRMT1b, which methylates H4R3, was up-regulated in all three NO-deficient

543

mutants (157). Another example is PRMT5, which catalyze symmetric di-methylation of

544

H4R3 in vitro and is essential for proper pre-mRNA splicing (158). PRMT5 was upegulated

545

in noa1-2 and nia1nia2noa1-2 44 and positively regulated by S-nitrosation during stress

546

responses (159). Regarding the late flowering phenotype of these NO-deficient mutants, it is

547

worth mentioning that JMJ18 was downregulated in each mutant. JMJ18 is a H3K4

548

demethylase, which controls flowering time. Enhanced endogenous levels of NO or GSNO,

549

due to overexpression of rat neuronal NOS (nNOS) or knockout of GSNOR, respectively,

550

resulted in downregulation of JMJ30 (151, 152). JMJ30 demethylates H3K36me2/3, regulates

551

period length in the circadian clock (160), and is involved in the control of flowering time. In

552

sum, the expression of DNA and histone methylation modifying enzymes is differentially

553

controlled by NO. This implies an indirect effect of NO on epigenetic mechanisms in plants.

554

Clearly, these observations are still very preliminary, as expression patterns from different

555

treatments are not consistent and partially contradictory, and verification of the resulting

556

DNA and histone methylation patterns is still lacking. The complexity is further illustrated by

557

a study in rice (Orzya sativa L. spp. Japonica), where high concentrations of the NO-donor

558

sodium nitroprusside induced global DNA hypomethylation (mainly in CHG sites) and led to

559

altered expression of chromatin remodeling enzymes (161).

560

Although proteome-wide studies identified several proteins involved in DNA and

561

histone methylation as candidates for S-nitrosation, functional analyses of these S-nitrosated

562

proteins are rare. Only S-nitrosation of PRMT5 has been described in detail (159). Briefly, S-

563

nitrosation of PRMT5 in Arabidopsis promotes its methyltransferase activity, which enables

564

methylation-dependent pre-RNA splicing associated with salt stress tolerance (159). Further,

565

ARGONAUTE 4 (AGO4), a component of the canonical RdDM pathway, was identified as

566

putative S-nitrosated in a proteome-wide approach (118). Based on the studies on human 17

567

JHDM (139), Fe(II)-dependent plant JHMDs might be targets for metal nitrosation by the

568

formation of a nitrosyl-iron complex with the non-heme Fe(II) in their catalytic pocket.

569

Hence, NO-induced changes of histone methylation by direct inhibition of the catalytic

570

activity of plant JHMDs is suggested. In regard, that iron-sulfur clusters of proteins are

571

targeted by NO resulting in the disruption of the cofactor, the iron-sulfur containing

572

ROS1/DME DNA demethylases might be affected by NO in plants.

573 574

4.4.

NO affects metaboloepigenetic processes interacting with histone and DNA

575

methylation

576

Intriguingly, several proteome-wide studies in plants revealed key enzymes of the

577

methylation cycle as targets for S-nitrosation, namely MS, SAMS, SAHH (117, 162-167), and

578

for tyrosine nitration, namely MS and SAHH (168-170) suggesting that NO impairs the

579

methylation cycle. In mammals, NO inhibits cobalamin-dependent MS due to its reaction

580

with the cofactor cobalamin (171-173). Unlike mammals, plants utilize solely cobalamin-

581

independent MS, which are identified as targets for S-nitrosation (117, 167) and nitration

582

(168). In Arabidopsis SAMS isoforms are differentially inhibited by protein S-nitrosation

583

(166). While SAMS1 is reversibly inhibited by GSNO, SAMS2 and SAMS3 are not affected.

584

It was demonstrated, that S-nitrosation of the Cys114 of SAMS1, which is located next to the

585

catalytic center as part of the active site loop, is responsible for inhibition (166). A similar

586

differential regulation of SAMS activity was observed in mammals. Here two genes encode

587

different SAMS isoforms. Only the isoform SAMS1A, but not SAMS2A was reversibly

588

inactivated by NO (174). Additionally, tyrosine nitration has been observed in SAHH of

589

sunflower causing a decreased SAHH activity (170). Further, site-specific S-nitrosation of

590

SAHH1 protein upon cold stress was reported, but the physiological consequence of cold

591

stress induced S-nitrosation of SAHH1 is not yet investigated (119, 175). Noteworthy, O-

592

acetylserine(thiol)-lyase is a target for S-nitrosation (117) and is inhibited by the nitration of

593

the tyrosine residue (176, 177). This enzyme is essential for the biosynthesis of cysteine and

594

methionine (178), which is converted to SAM in the methylation cycle. Taken together, NO

595

may play a regulatory role in different enzyme activities for the synthesis of sulfur-containing

596

amino acids and the methylation cycle providing the methyl-group donor SAM for

597

transmethylation reactions. In addition to the identification of proteins, which are targets for

598

NO-induced PTMs, the effect of NO on metabolic reprogramming using untargeted and

599

targeted metabolomics together with transcriptomic profiling is an emerging field. In this

600

regard, transcriptomic profiling upon NO-donor treatment and in mutants with altered NO 18

601

homeostasis revealed that genes involved in methyl donor supply are regulated by NO. For

602

instance, treatment of Arabidopsis cell suspension with 0.5 mM NOR3 resulted in

603

downregulation of MS1, SAMS3, and SAMS4. In contrast, exposure of 4-5 week old

604

Arabidopsis plants to gaseous NO resulted in an induction of SAMS4 (179). Moreover,

605

CysNO treatment induced expression of SAMS2, SAMS3, SAMS4, SAHH1, and SAHH2. In

606

addition, genes involved in folate, cysteine, methionine, and GSH biosynthesis, and

607

methylation cycle were differentially regulated upon CysNO treatment (148). Transcriptomic

608

profiling of NO-deficient mutants noa1-2, nia1nia2, and nia1nia2noa1-2 revealed that genes

609

coding for enzymes involved in the methylation cycle, folate, cysteine, methionine, and GSH

610

biosynthesis are differentially regulated (151). Recently, an untargeted metabolomic analysis

611

revealed that NO affect glutathione, methionine, and carbohydrate metabolism in elm seeds

612

(180). In particular, SNP and GSNO modulate the methylation cycle at the transcriptional

613

level by elevating SAMS transcript levels and by increasing the levels of methionine and

614

SAM (180). In sum, these data demonstrate that NO affect the methyl-donor supply by

615

altering transcription of genes coding for enzymes involved in SAM biosynthesis, changing

616

enzyme activities, and altering metabolic levels.

617

Transcriptomic profiling also revealed numerous genes involved in the TCA cycle that

618

are differentially expressed upon CysNO treatment or in NO-deficient mutants (noa1-2,

619

nia1nia2, nia1nia2noa1-2) (148, 155). For instance, ACONITASE 2 and 3 (ACO2, ACO3)

620

were upregulated in nia1nia2 and nia1nia2noa1-2. Further, it was demonstrated that NO

621

inhibits aconitase by forming a metal-nitrosyl complex with its iron-sulfur cluster (181, 182).

622

In addition, ACO was found as target for S-nitrosation (162, 183). It is known that under

623

stress conditions NO regulate the cellular redox state, which might influence the availability

624

of FAD and thus the activity of histone demethylase LSD1 (184). Further, untargeted

625

metabolic analysis revealed that plants exposed to NO for six hours undergo a transient

626

metabolic reprogramming, including increased succinate (inhibit JHDMs) and decreased α-

627

KG (substrate of JHDMs) levels (115). Both NO-deficient mutants nia1nia2 and

628

nia1nia2noa1-2 display an impaired TCA cycle. The nia1nia2noa1-2 mutant plants displays a

629

significant increased level of succinate and a decreased level of fumarate (inhibit JHDMs),

630

whereas the α-KG was not altered compared to wild-type (185). In contrast, the succinate and

631

fumarate level were decreased in the nia1nia2 mutant (186). Hence, it is suggested that NO

632

may regulate histone methylation through effecting these metabolites. Taken together, NO is

633

supposed to be an epigenetic regulator of DNA and histone methylation in plants based on S-

19

634

nitrosoproteomic studies, transcriptomic profiling, and metabolic analysis using exogenous

635

NO-donor treatments and mutants with impaired NO homeostasis.

636 637 638 639

5.

Interactions of redox-signaling, metabolism, and chromatin modification in

640

response to environmental stress

641

The ability to adjust cellular programs to the environmental conditions seems

642

particularly pronounced in plants (187). Plants are generally sessile and therefore cannot

643

evade biotic and abiotic stresses. Perennial plants are challenged by seasonal fluctuations and

644

long-lived tree species even face climate-change phenomena during their life time. Epigenetic

645

mechanisms have been postulated to confer phenotypic plasticity and stress memory in plants.

646

Although environmental responses are often temporary, life-threatening events, such as heat

647

or pathogen attack, can elicit systemic responses and sustained morphological and

648

physiological provisions for recurring stress that are known as acclimation or priming (188).

649

Enhanced stress tolerance may even be inherited, but in most examples transgenerational

650

stress memory is erases within one stress-free generation (34, 189, 190). Nevertheless, the

651

notion that epigenetic mechanisms are at the core of stress memory has been substantiated by

652

studies showing that specific histone modifications, in particular histone H3K4 di- and tri-

653

methylation, and chromatin remodeling factors are involved in the priming of stress response

654

genes (191, 192). Importantly, chromatin modifications can thereby act as a memory and lead

655

to faster or stronger gene responses during subsequent stress (189, 190, 193).

656

By now, numerous methylome studies in Arabidoposis, rice and other plant species

657

have shown altered DNA methylation patterns upon different environmental challenges,

658

including phosphate starvation, hyperosmotic stress, chemical elicitors and pathogen

659

treatment (190, 194-196). For example, the bacterial pathogen Pseudomonas syringae pv.

660

tomato (Pst) as well as hyperosmotic stress lead to sizeable changes in somatic DNA

661

methylation patterns in the challenged plants and the direct progeny (190, 194, 195).

662

Moreover, increased resistance to bacterial pathogens as well as hyperosmotic stress in the

663

progeny is associated with specific DNA methylation patterns and lost in DNA methylation

664

and DNA glycosylase mutants, indicating that DNA methylation changes at RdDM target

665

sites might act as stress memory and control the inducibility of response genes (190, 197).

666

Although expression changes of RdDM genes and DNA glycosylases, as observed

667

upon Pst treatment, might account for some of these DNA methylation dynamics (193), the 20

668

involved mechanisms are not well understood. In contrast, it is well known that biotic and

669

abiotic stress leads to profound metabolic responses that are commonly characterized by an

670

immediate burst in ROS production, and often followed by redox-mediated signaling

671

mechanisms (6, 198, 199). Based on the outlined reports on thiol-based redox-regulated

672

nuclear proteins, there is increasing support for the concept that redox- -mediated signaling

673

affect the epigenetic regulation by changes in the activity of histone and DNA modifying

674

enzymes (Figure 2). In this context, it is also noteworthy that oxidative stress has been

675

implicated with human pathologies that are connected to altered DNA methylation, particularly cancer

676

(200). ROS/NO may directly affect the activity of chromatin modifiers or regulate the supply

677

of methyl group donors and methylation inhibitors via redox-dependent protein modifications

678

(Figure 2). Production of primary and secondary plant metabolites that depend on SAM and

679

act as protectants, detoxification agents, or hormones, such as betaine glycine, spermidine,

680

and ethylene, can moreover lead to increased C1 demands upon stress and thereby to

681

competition for potentially limited folate pools (70, 201). Due to the described sensitivity of

682

DNA and histone methyltransferases as well as DNA glycosylases to NO signaling, redox,

683

and C1 homeostasis, it is therefore possible that metabolic shifts can cause enhanced or

684

reduced chromatin methylation in response to environmental cues. In line with this

685

hypothesis, folate-mediated changes in DNA methylation were recent linked to altered

686

pathogen resistance towards Pst (76). Using a chemical genetic approach, the authors showed

687

that antifolates, which are known block the methionine cycle and thereby interfere with DNA

688

methylation in plants, enhance resistance against Pst, whereas overexpression of MS1 led to

689

DNA hypermethylation, particularly in the non-CG context, and attenuated resistance (76).

690

Metabolites, redox processes and chromatin structure are important key factors to regulate

691

gene expression and to achieve a rapid, effective and appropriate response to a changing

692

environment a coordination of epigenetics, metabolism and redox events is essential for plant

693

cells. However, whether, how and under which circumstances distinct metabolic changes

694

impact chromatin structure or vice versa still needs to be investigated in more detail. Some

695

examples about the direct function of metabolites/redox-compounds on chromatin

696

modification are already reported. For instance, an increase in acetyl-CoA is accompanied by

697

an increase in H3K27ac resulting in changes α-ketoglutarate levels (27). α-Ketoglutarate in

698

turn is an important metabolic intermediate acting as a cofactor for several chromatin-

699

modifying enzymes (28) demonstrating on one side how primary metabolism specifically can

700

affect chromatin structure and on the other side how these chromatin changes affect

701

accumulation of specific metabolites, which in turn can affect chromatin modulation (27). 21

702

This shows at least that the interplay of epigenetics and metabolites/redox-compounds is bi-

703

directional, meaning that the chromatin structure of metabolic- or redox-related genes is

704

important to define the accessibility of these genes for the transcription machinery and on the

705

other hand that distinct metabolites or redox-compounds are directly involved in the

706

regulation of chromatin modifications. However, fundamental questions regarding the how

707

metabolites affect chromatin modification and gene expression remain. E. g. which chromatin

708

modifiers preferentially respond to fluctuations in metabolite levels and which chromatin

709

modifications are involved regulating transcription of metabolic- and redox-related genes and

710

accumulation of metabolites and redox-compounds? Time-course studies and analysis of ko-

711

mutants of genes involved in metabolite synthesis and redox molecule production would be

712

promising approaches to identify the reaction processes and networks between chromatin

713

modification, gene expression, and metabolites/redox processes.

714 715 716

6.

Conclusions and outlook

717

Several proteins involved in primary or secondary metabolism also participate to

718

chromatin modulation and gene regulation by providing co-factors or substrates for epigenetic

719

modifiers highlighting a strong interconnection of metabolism and epigenetics. However, our

720

understanding of the detailed function of metabolites in epigenetic regulation during stress

721

responses is still in its infancy. One of the challenges in the field is the dissection of the many

722

pleiotropic ramifications of metabolic regulation in order to distinguish between direct and

723

indirect effects. Experiments using NO donor treatments have shown some interesting

724

expression changes in genes encoding known chromatin-modifying proteins, some of which

725

are known to be redox sensitive in animals. Yet, complementary analyses of chromatin

726

modification patterns are missing in most cases, and even if present, the causalities are

727

difficult to determine, as chromatin modification and transcriptional dynamics often go hand-

728

in-hand. One approach that has been used successfully to describe redox regulation

729

mechanism in other biological contexts, is based on the identification of SNO and other

730

redox-dependent PTM targets by proteomics. Following their identification, in vitro and in

731

vivo functional characterization of their redox-dependent activities, substrates and/or

732

interaction partners can provide valuable new insights into the signaling of environmental

733

cues to the chromatin level.

734

By now, it is well established in animals as well as in plants that the levels of

735

metabolic intermediates often correlate globally with the levels of the epigenetic 22

736

modifications, for which they serve as a cofactor. Yet, the biological implications, such as

737

connections between diurnal metabolic changes and the circadian clock, are largely unknown

738

in plants. One the one hand,

739

we still need a better picture of the dynamics, tissue specificities and subcellular distributions

740

of the involved metabolites, including folate species and thiols, which also requires further

741

development of the quantification methods. One the other hand, we have to address the

742

problem to what extent and how cofactors levels are involved in the regulation of specific

743

chromatin modification pathways or certain genomic regions. Regarding histone methylation,

744

different studies in Arabidopsis indicate that DNA methylation and H3K9me2 are particularly

745

sensitive to changes in SAM and/or SAH levels (9, 202), and the likely connection to the

746

feedback mechanism between those two chromatin marks has been discussed. In comparison,

747

reduced SAM and SAH levels due to Met restriction in human cells mostly affected

748

H3K4me3 (203). It is known that histone methyltransferases differ in their binding affinities

749

towards SAM and SAH (204), which also might lead to different sensitivities of the

750

corresponding methylation marks towards metabolic fluctuations. Furthermore, different

751

global and regional dynamics and turnover rates of chromatin marks and nucleosomes can

752

also affect cofactor dependencies. Another mechanism to provide specificity for different

753

pathways as well as certain genomic regions constitutes metabolic channeling through

754

enzyme complexes. In mammals and yeast, recent studies suggest that nuclear components of

755

the methionine cycle are involved in epigenetic regulation by directly interacting with histone

756

methyltransferases. For example, it has been shown that the catalytic subunit of mammalian

757

SAM synthetase, MATIIα, is recruited to loci involved in oxidative stress and immune

758

response, where it interacts with different chromatin-associated proteins, including the histone

759

methyltransferases SETDB1 and SUV39H1, to provide SAM for H3K9me3 (205).

760

Remarkably, MATIIα activity is not required for H3K4me3 at these loci. In yeast, it has been

761

shown that SAM synthetase associates with a chromatin-modifying complex, which includes

762

metabolic enzymes and thereby may coordinate metabolic state with epigenetic regulation

763

(206). Such complexes have not yet been described in plants but, intriguingly, plant nuclei

764

show defined folate metabolic activities and many proteins involved folate-mediated C1

765

metabolism are localized in the cytosol and the nucleus (80, 207). For some of these proteins,

766

nuclear methylation functions have been proposed (208, 209), but their roles in chromatin

767

dynamics and epigenetic regulation remain elusive.

768

It is noteworthy that several pathways involving methionine cycle intermediates are

769

not conserved between plants and animals, why many genes associated with elevated plasma 23

770

Hcy in humans do not have homologs in plants (e.g. GNMT, CBS, and ALDH1L1) (210). In

771

addition to what is known, plants might have evolved alternative ways to control Hcy and

772

SAH levels. Their steady-state-levels seem to be tightly controlled (211) and affected by

773

environmental factors, e.g. nutrient availability (212). The mechanisms controlling Hcy and

774

SAH levels have not been investigated thoroughly in plants, but due to the sensitivity of DNA

775

and histone methylation to changes in SAM/SAH levels they might include yet unknown

776

factors involved in chromatin methylation dynamics.

777

Another challenge is the separation of genetic and epigenetic effects. To this end, the

778

use of epigenetic recombinant inbred lines (epiRILs) in Arabidopsis has been seminal, leading

779

to the identification of DNA methylation-dependent quantitative traits (213). Further analyses

780

along those lines with particular focus on metabolic traits may lead to the identification of

781

new mechanisms involved in epigenetic regulation. Finally, the identified mechanisms have to

782

be analyzed in a developmental context and the natural environment to complement the

783

picture of ROS/NO signaling, metabolism, and epigenetic functions in plants. Large-scale

784

analyses of natural genetic and epigenetic variation in Arabidopsis linked changes in DNA

785

methylation to environmental adaptation (214-216). The tracking of epigenetic variation over

786

multiple generations under environmental simulation of projected climate parameters,

787

including CO2 levels, temperature, and soil water content, will be instrumental to define long-

788

term environmental effects on epigenetic regulation and the possible impact of global

789

warming on chromatin modification patterns. As stated initially, our overarching interested in

790

the interactions between metabolism and chromatin lies in the environmental impact on

791

epigenetics, which is relevant from the health, agricultural, and ecological perspective, alike.

792

The unraveling of these interactions will require interdisciplinary approaches and can greatly

793

benefit from the use of different model systems, including plants, as hopefully conveyed by

794

this review.

795 796 797

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Figures and Tables

1355 1356

1357 1358 1359 1360 1361 1362 1363 1364 1365

Figure 1

Figure 1: Folate-mediated C1 metabolism in plants and its involvement in DNA and histone methylation. Identified mutations that affect DNA/histone methylation are highlighted in red (THF in red letters refers to THF polyglutamylation by plastidic FPGS1; description and citations in main text). (1) serine hydroxymethyltransferase (SHMT),

(2)

methylenetetrahyrofolate

reductase

(MTHFR),

methionine

synthase

(MS),

(4a)

formyltetrahyrofolate synthetase (FTHFS), (4b) MTHFD1 - cyclohydrolase, (4c) MTHFD1 - dehydrogenase, (5) SAM synthetase (SAMS), (6) SAM-dependent methyltransferases, (7) SAH hydrolase (SAHH), (8) glycine decarboxylase complex (GDC), (9) 5-formyltetrahyrofolate cycloligase, (?) putative mitochondrial folate transporter.

1366 1367

(3)

Figure 2

35

1368 1369 1370 1371 1372 1373 1374 1375

Figure 2. Model for stress interactions of metabolism and chromatin modification. Environmental stress leads to increased production of reactive oxygen species (ROS) and NO. ROS can lead to inactivation of DNA glycosylases involved in DNA demethylation (e.g. ROS1). NO acts as an HDAC inhibitor and may also affect the activity of enzymes in the methionine cycle, e.g. SAMS4 (1) and SAHH1 (2), leading to changes in SAM and SAH levels. This can lead to changes in histone lysine methylation (Kme) and cytosine methylation (5mC), as DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs) require SAM as methyl donor and are inhibited by SAH. Moreover, the methionione cycle depends on C1 supply from the folate cycle.

1376 1377 1378 1379

Table: NO-induced changes in expression of genes related to histone and DNA methylation. Previously reported transcriptomic analyses were screened for differentially expressed genes involved in histone and DNA methylation pathways. Treatment (Citation) Infiltration of 4week-old leaves with 1 mM CysNO

Up-/downregulated Up

Down

Microarray analysis WT vs. noa1-2, decreased NO level

Up

Down

Microarray

Up

Gene

Function/pathway

STABILIZED1, RDM16, HSP90-1 CMT2 AGDP1 SUVR3/SDG20 JMJ13, JMJ21, JMJ26, JMJ29 ROS1, DML2, IDM3 NRPD2/NRPE2, NRPE5, AGO4, DMS3/IDN1, KTF1, IDP1, IDP2, SUVH9, LDL1, RRP6L1 DDM1 SWN/SDG10, SHR3/SDG4, ATX5/SDG29, SUVH9/SDG22, SUVR2/SDG18, PRMT4A JMJ27, LDL1 DML2 NRPD4/NRPE4, NRPE5, HSP90-1 MET1, VIM1 ATXR7/SDG2, PRMT1a, PRMT1b, PRMT3, PRMT10, PRMT5, JMJ22 CMT2 ASHH3/SDG7, SUVR2/SDG18 JMJ18 NBP35 RDM1

RdDM CHG/CHH methylation AGDP1 linking DNA and H3K9me2 Histone methylation Histone demethylation DNA demethylation RdDM

36

DNA methylation Histone methylation

Histone demethylation DNA demethylation RdDM CG methylation Histone methylation

CHH methylation Histone methylation Histone demethylation CIA pathway RdDM

analysis WT vs. nia1nia2, decreased NO level

Microarray analysis WT vs. nia1nia2noa1-2, decreased NO level

Down

Up

Down

PRMT1b, PRMT3, PRMT4B ROS1, DML2 DCL3, DRD1, IDN1, LDL1, BP26, RRP6L1, kRDM16 MET1, CMT2 ASHH3/SDG7, ATXR6/SDG34, SUVH1/SDG32, JMJ11, JMJ18, JMJ28 APE1L RDM1 PRMT1b, PRMT3, PRMT4B, RMT10, PRMT5 NAR1, DRE2, NBP35, CIA1 ROS1, MBD7 AGO4 CMT2 ATX5/SDG29, ATXR6/SDG34, UVH6/SDG23, SUVR2/SDG18 JMJ18

1380 1381 1382

37

Histone methylation DNA demethylation RdDM DNA methylation Histone methylation Histone demethylation DNA demethylation RdDM Histone methylation CIA pathway DNA demethylation RdDM CHH methylation Histone methylation Histone demethylation

Highlights

• • • •

Plant specific non-CG methylation pathways are particularly sensitive to changes in folate-mediated one-carbon metabolism. Reactive oxygen species (ROS) and nitric oxide (NO) in coordinate metabolic pathways and gene activities that are involved in chromatin modulation. As ROS and NO are hallmarks of stress responses, they might also pivotal in mediating chromatin dynamics during environmental stress responses. Mammals and plants share a common dependence on metabolic intermediates that serve as cofactors for DNA/histone methylation, histone demethylation, and histone acetylation.

1

Conflict of interest statement

Interaction between metabolism and chromatin in plant models Christian Lindermayr, Eva Esther Rudolf, Jörg Durner, and Martin Groth

NO conflict of interest.