Epigenetic regulation of sulphur homeostasis in plants

Highlight statement: We summarize and discuss recent findings on the epigenetic 11 regulation of sulphur homeostasis and response to sulphur deficiency in plants, including 12 DNA methylation, histone modifications and noncoding RNA mediated gene silencing. Abstract 30 Plants have evolved sophisticated mechanisms for adaptation to fluctuating availability of 31 nutrients in soil. Such mechanisms are of importance for plants to maintain homeostasis of 32 nutrient elements for their development and growth. The molecular mechanisms 33 controlling the homeostasis of nutrient elements at the genetic level have been gradually 34 revealed, including the identification of regulatory factors and transporters responding to 35 nutrient stresses. Recent studies have suggested that such responses are not only controlled 36 by genetic regulation but also by epigenetic regulation. In this review, we present recent 37 studies on the involvement of DNA methylation, histone modifications and noncoding 38 RNA mediated gene silencing in the regulation of sulphur homeostasis and response to 39 sulphur deficiency. We also discuss the potential effect of sulphur containing metabolites 40 such as S -adenosylmethionine (SAM) on the maintenance of DNA and histone methylation. 41 42 enzymes in red change histone methylation. Abbreviations for enzymes: ADK, adenosine kinase; APS reductase; sulfurylase; cystathionine β-lyase; synthetase. for compounds: adenosine adenosine

The word count (start of the introduction to the end of the acknowledgements): 5891 29 Introduction to activate sulphate uptake in roots, and SULTR4;2 to release sulphate from vacuoles 76 (Maruyama-Nakashita et al., 2006). Several cis-elements responsive to S deficiency have 77 been identified, including the sulphur-responsive element (SURE) in the promoter of 78 SULTR1;1 (Maruyama-Nakashita et al., 2005), a SURE-like element in the promoter of 79 the wheat Sulfur deficiency-induced-1 (sdi-1) gene (Howarth et al., 2009), the UPE-box in 80 tobacco UP9C gene (Wawrzynska et al., 2010), and SURE21A and SURE21B in the 3'-81 untranslated region of SULTR2;1 (Maruyama-Nakashita et al., 2015). It appears that 82 SLIM1 does not target directly to the SURE element in the promoter of SULTR1;1 and 83 SULTR1;2 though it regulates the expression of these two gens. Rather, SLIM1 forms a 84 homodimer and binds to the UPE-box, which also exists in the promoters of sulphur 85 deficiency induced genes in Arabidopsis, such LSU, APR and SULTR2;1 (Wawrzynska et 86 al., 2010;Wawrzynska and Sirko, 2016). 87 Similar to the complex regulation of sulphate uptake and distribution, sulphate assimilation 88 is also tightly controlled, being highly regulated by the demand for reduced sulphur, in a 89 regulatory system known as the 'demand-driven' regulatory pathway (Davidian and 90 Kopriva, 2010;Lappartient and Touraine, 1996;Lappartient et al., 1999). However, the 91 molecular mechanisms underlying the regulation of sulphate assimilation remain largely 92 unclear. SLIM1 is likely involved in regulating the expression of ATPS4 and SERAT3;1 as 93 these two genes are downregulated in the slim1 mutant (Maruyama-Nakashita et al., 2006). 94 The transcriptional factor LONG HYPOCOTYL5 (HY5) has been shown to regulate the 95 expression of APR1 and APR2 in Arabidopsis by directly targeting the promoters of these 96 two genes (Lee et al., 2011). However, HY5 seems to not regulate the expression of APR3, 97 suggesting multiple genetic pathways for the regulation of the reduction of APS. The 98 regulation of the biosynthesis of sulphur containing secondary metabolites such as 99 glucosinolates is much more complex. Many transcription factors, including at least eight 100 MYBs, six MYC-bHLHs, two WRKYs, and a DNA-binding-with-one-finger (DOF) 101 transcription factor OBP2, have been shown to be involved in this process (Frerigmann,102 2016). Recently, two repressors controlling glucosinolate biosynthesis, sulfur deficiency 103 induced 1 (SDI1) and SDI2 have been identified in Arabidopsis (Aarabi et al., 2016). Under 104 sulphur limited conditions the nuclear localized SDI1 interacts with MYB28, a major 105 transcription factor that promotes glucosinolate biosynthesis, to suppress the biosynthesis of glucosinolates and prioritize sulphate utilisation for primary metabolites (Aarabi et al.,107 2016). The catabolic recycling of organic S compounds such as glucosinolates and GSH is 108 essential for plants to adapt to sulphur limiting conditions. Glucosinolates are thought to 109 function as a sulphur storage pool in plants in the Brassicaceae as their levels fluctuate 110 according to the environmental sulphur status (Falk et al., 2007;Maruyama-Nakashita, 111 2017; Maruyama-Nakashita et al., 2006). Although the catabolic enzymes of 112 glucosinolates and GSH have been identified and well characterized (Bachhawat and 113 Yadav, 2018;Kumar et al., 2012;Kumar et al., 2015;Ohkama-Ohtsu et al., 2008;Paulose 114 et al., 2013;Wittstock and Burow, 2010), the genetic regulation of the breakdown of these 115 compounds is largely unknown. Except SLIM1 which functions as a central transcriptional 116 regulator in the degradation of glucosinolates under sulphur limited conditions 117 (Maruyama-Nakashita et al., 2006), other transcription factors and corresponding targeting 118 cis-elements involved in the degradation of glucosinolates and GSH remain to be identified.

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It is well recognized that the regulation of S homeostasis is under complex genetic control.

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Emerging evidence suggests that epigenetic regulation of gene expression plays an 121 important role in the adaptive response to S deficiency and the maintenance of S 122 homeostasis (Huang et al., 2016). Epigenetic changes refer to heritable genetic changes 123 resulting from modification of a chromosome without alteration of the DNA sequence 124 (Berger et al., 2009). Epigenetic regulation of gene expression in response to biotic and 125 abiotic stresses, and adaptation to environmental cues, has been gradually revealed (Alonso 126 et al., 2019;Chinnusamy and Zhu, 2009;Lamke and Baurle, 2017;Sahu et al., 2013;Secco 127 et al., 2017). Epigenetic regulation mainly occurs at three levels; DNA methylation, histone 128 modifications, and noncoding RNA regulation. Perhaps the most direct link between S 129 homeostasis and DNA and histone methylation is the fact that S-adenosylmethionine 130 (SAM), a major methyl donor required for many transmethylation reactions, is a sulphur 131 containing compound. In this review, we discuss what is currently known about the 132 regulation of S homeostasis at these three epigenetic levels.  Arabidopsis under phosphate starvation (Secco et al., 2015), this may be due to different 173 treatment conditions and/or different approaches in the identification of differentially 174 methylated regions (Secco et al., 2017). Zinc deficiency also triggers genome-wide  Under sulphate deficient condition, SAM concentration decreases (Nikiforova et al., 2005). 190 Recently, using BS-seq to investigate genome-wide changes in DNA methylation in 191 response to sulphur deficiency, we observed that cytosine methylation levels in all three 192 sequence contexts CG, CHG and CHH decreased in both roots and shoots under sulphate 193 depletion conditions ( Fig. 2A). This might be due to a shortage of the methyl donor SAM 194 which potentially lead to enhanced passive DNA demethylation (Zhang et al., 2018). to SAH (Sauter et al., 2013). The SAM to SAH ratio is generally termed the 'methylation 211 potential' and can be used as a metabolic indicator for the methylation status in cells. The 212 alteration of the SAM to SAH ratio usually leads to changes in global methylation patterns.

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Partial loss-of-function of SAHH1 (also known as HOMOLOGY-DEPENDENT GENE 214 SILENCING1, HOG1) leads to increased SAH levels and a decreased SAM to SAH ratio 215 resulting in DNA hypomethylation in Arabidopsis (Ouyang et al., 2012;Rocha et al., 2005).

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A subset of genes is up-regulated in the hypomethylated hog1 mutant, which shows a 217 dramatic growth defect (Jordan et al., 2007;Rocha et al., 2005). Reduction of ADK activity leading to the activation gene expression (Fig. 3). .

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Although there is no direct evidence to support histone modifications involvement in 376 regulation of sulphur homeostasis, histone methylations and acetylations are found in many 377 genes involved in sulphate uptake and assimilation in Arabidopsis (Table 1) , and H3K9ac (Zhou et al., 2010). Therefore, it can be 380 assumed that histone modification may also play a role in maintaining sulphur homeostasis.
In fact, the interruption of the SAM cycle, which leads to abnormal SAM to SAH ratio, 382 affects histone methylation (Fig. 1). Mutations of FPGS1, MTHFD1 and SAMS3, which all 383 lead to lower SAM to SAH ratios, not only reduce global DNA methylation but also 384 decrease H3K9me2 levels (Groth et al., 2016;Meng et al., 2018;Zhou et al., 2013).

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Furthermore, elevation of SAH has been shown to decrease the methylation of histone H3 and in some cases also changes in histone methylation (Fig. 1). Therefore, a tight link 486 between sulphur metabolism and DNA and histone methylation appears to exist in plants. 487 Indeed, mutation of MSA1/SHM7 leads to a reduction of SAM levels and alters global DNA    γ-GluCys, γ-glutamylcysteine.  Whole genome analysis of histone modifications was carried out by using chromatin immunoprecipitation (ChIP) coupled with high-density whole genome tiling microarrays (ChIP-chip), or ChIP coupled with high throughput sequencing (ChIP-seq). Genes involved in sulphate uptake and assimilation were extracted and shown in Table 1 Fig. 1. The interconnection of sulfate assimilation, folate metabolism and the SAM cycle with the DNA and histone methylation. The sulfate uptake and assimilation pathway, the biosynthesis and turnover of folate and the SAM cycle were shown in the background of light green, light blue and orange colors, respectively. Interruption of enzymes in blue and red colors alters genomewide DNA methylation, and mutation of enzymes in red color change histone methylation. Abbreviations for enzymes: ADK, adenosine kinase; APK, APS kinase; APR, APS reductase; ATPS, ATP sulfurylase; CBL, cystathionine β -lyase; CGS, cystathionine γ -synthase; DHFR, DHF reductase; DHFS, DHF synthase; DHPS, DHP synthase; γ -ECS, γ -glutamylcysteine synthetase; FPGS, folylpolyglutamate synthase; GSHS, glutathione synthetase; MS, methionine synthase; MTHFD1, bifunctional methylene THF dehydrogenase/methenyl THF cyclohydrolase; OAS-TL, OAS(thiol)lyase; SAHH, SAH hydrolase; SAMMT, SAM-dependent methyltransferase; SAMS, SAM synthetase; SAT, serine acetyltransferase; SHM, serine hydroxymethyltransferase; SiR, sulphite reductase; SOT, sulfotransferase; SULTR, sulfate transporter; SYN, 10-formyl THF synthetase. Abbreviations for compounds: Ado, adenosine; AMP, adenosine monophosphate; APS, adenosine 5'-phosphosulfate; Cys, cysteine; Cyst, cystathionine; DHF, dihydrofolate; DHP, dihydropteroate; Glun, polyglutamate; Hcy, homocysteine; Met, methionine; OAS, O-acetylserine; pABA, UDP-glucose-p-aminobenzoate ; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; Ser, serine; THF, tetrahydrofolate; γ -GluCys, γ -glutamylcysteine.  Fig. 2. Whole genome methylation level of Arabidopsis under sulfate and phosphate starvation conditions. (A) Methylation levels at all cytosines in the genome (Total C) and the CG, CHG and CHH sequence context under +S and -S conditions. Methylation level was determined by whole genome bisulfite sequencing (BS-Seq) on the shoots and roots of plants grown on MGRL agar media with 1.5 mM sulfate (+S) or without added sulfate (-S) for two weeks. (B) Methylation levels at all cytosines in the genome (Total C) and the CG, CHG and CHH sequence context under +Pi and -Pi conditions. Data were derived from Yong-Villalobos et al. (2015) and recalculated based on the raw data. Plants were grown hyponically with 1 mM phosphate for 7 days and then transferred to hydropic media containing 1 mM (+Pi) or 5 µM phosphate (-Pi) for 16 days. Methylation level was determined by BS-Seq on the shoots and roots, respectively.