Upload
others
View
5
Download
0
Embed Size (px)
Citation preview
1
METHIONINE ADENOSYLTRANSFERASE 4 mediates DNA and histone 1
methylation 2
Jingjing Meng#, Lishuan Wang#, Jingyi Wang, Xiaowen Zhao, Jinkui Cheng, 3
Wenxiang Yu, Dan Jin, Qing Li, and Zhizhong Gong* 4
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological 5
Sciences, China Agricultural University, Beijing 100193, China. 6
#These authors contribute equally to this work. 7
*Corresponding author: 8
Zhizhong Gong 9
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological 10
Sciences, China Agricultural University, Beijing 100193, China. 11
Email: [email protected]; Tel: 86-10-62733733 12
Key words: DNA methylation, histone methylation, gene silencing, Arabidopsis, 13
SAM 14
Author Contributions 15
Z. G conceived the original research plans; J. M. performed most of the experiments; 16
L.W. performed the mutant screening and gene cloning; J. C. provided bioinformatics 17
analysis; X. Z, J. W, D. J, W. Y and Q. L. assisted with some experiments. J. M. and Z. 18
G designed the project and wrote the article with contributions from all the authors. 19
Running title: MAT4 mediates DNA and histone methylation 20
One sentence summary: MAT4 is an essential gene in Arabidopsis that plays key 21
roles in regulating DNA and histone modifications as well as plant growth and 22
development. 23
24
Plant Physiology Preview. Published on March 23, 2018, as DOI:10.1104/pp.18.00183
Copyright 2018 by the American Society of Plant Biologists
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
2
Abstract 25
DNA and histone methylation co-regulate heterochromatin formation and gene 26
silencing in animals and plants. To identify factors involved in maintaining gene 27
silencing, we conducted a forward genetic screen for mutants that release the silenced 28
transgene Pro35S::NEOMYCIN PHOSPHOTRANSFERASE II in the transgenic 29
Arabidopsis thaliana line L119. We identified MAT4/SAMS3/MTO3/AT3G17390, 30
which encodes methionine adenosyltransferase 4 (MAT4)/S-adenosyl-methionine 31
synthetase 3 that catalyzes the synthesis of S-adenosyl-methionine (SAM) in the 32
one-carbon metabolism cycle. mat4 mostly decreases CHG and CHH DNA 33
methylation and histone H3K9me2 and reactivates certain silenced transposons. The 34
exogenous addition of SAM partially rescues the epigenetic defects of mat4. SAM 35
content and DNA methylation were reduced more in mat4 than in three other mat 36
mutants. MAT4 knock-out mutations generated by CRISPR/Cas9 were lethal, 37
indicating that MAT4 is an essential gene in Arabidopsis. MAT1, 2, and 4 proteins 38
exhibited nearly equal activity in an in vitro assay, whereas MAT3 exhibited higher 39
activity. The native MAT4 promoter driving MAT1, 2 and 3 cDNA complemented the 40
mat4 mutant. However, most mat4 transgenic lines carrying native MAT1, 2, and 3 41
promoters driving MAT4 cDNA did not complement the mat4 mutant, because of 42
their lower expression in seedlings. Genetic analyses indicated that the mat1mat4 43
double mutant is dwarfed and the mat2mat4 double mutant was non-viable, while 44
mat1mat2 showed normal growth and fertility. These results indicate that MAT4 45
plays a predominant role in SAM production, plant growth and development. Our 46
findings provide direct evidence of the cooperative actions between metabolism and 47
epigenetic regulation. 48
49
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
3
INTRODUCTION 50
DNA and histone methylation are important epigenetic modifications that regulate 51
gene expression and genome stability, and can be inherited (Law and Jacobsen, 2010). 52
Compared with animals, which largely display CG methylation, plants present 53
symmetric CG and CHG methylation and asymmetric CHH methylation. In 54
Arabidopsis (Arabidopsis thaliana), DNA methylation is mediated by the 55
RNA-directed DNA methylation pathway (RdDM) and dicer-independent RdDM 56
(Matzke et al., 2015; Yang et al., 2016; Ye et al., 2016); CG methylation is maintained 57
by DNA METHYLTRANSFERASE1 (MET1, a functional equivalent protein of DNA 58
methyltransferase 1 in mammals); CHH methylation is maintained by DOMAINS 59
REARRANGED METHYLASE 2 (DRM2) and CHROMOMETHYLASE2 (CMT2) 60
(Cao and Jacobsen, 2002, 2002; Du et al., 2014); and CHG methylation is maintained 61
by CMT3. CHG methylation is recognized by the SET and RING Associated (SRA) 62
domain histone methyltransferase KRYPTONITE/(SU(VAR) homolog (SUVH)4 63
(KYP/SUVH4), and its homologs SUVH5 and SUVH6 to establish the dimethylation 64
of histone H3 at lysine 9 (H3K9me2) (Jackson et al., 2002; Ebbs et al., 2005; Ebbs 65
and Bender, 2006). H3K9me2 is bound by CMT3 through its H3 tails (Johnson et al., 66
2007; Bernatavichute et al., 2008; Law and Jacobsen, 2010; Du et al., 2012), which 67
form a reinforcing feedback loop that maintains CHG methylation and H3K9me2. 68
The one-carbon metabolism pathway plays an important role in epigenetic 69
regulation because it provides methyl groups for most methylation reactions (Fig. 1). 70
The initial methyl group donor is polyglutamate-5-methyl-tetrahydrofolate 71
(5-CH3-THF-Glun), which is the most common form of folate and has a high affinity 72
for folate-dependent methionine synthase as the methyl-group donor. 73
Folate-dependent methionine synthase catalyzes the methylation of homocysteine to 74
methionine using 5-CH3-THF-Glun as the methyl-group donor (Friso et al., 2002; 75
Ravanel et al., 2004; Mehrshahi et al., 2010). S-adenosyl-methionine (SAM), one of 76
the most abundant co-factors in plant metabolism, is synthesized by methionine 77
adenosyltransferase (MAT) (also known as S-adenosyl-methionine synthetase, SAMS) 78
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
4
using methionine and ATP as substrates. After transferring a methyl group to DNA, 79
RNA, proteins or other metabolites by SAM-dependent methyltransferases (Sauter et 80
al., 2013), SAM is changed into S-adenosyl-homocysteine (SAH), which competes 81
with SAM and is an inhibitor for many Methionine Synthetases (MTs) (Molloy, 2012). 82
SAH is then converted to adenosine and homocysteine by SAH hydrolase encoded by 83
the HOMOLOGY-DEPENDENT GENE SILENCING1 (HOG1) in Arabidopsis, thus 84
finishing a single cycle of one-carbon metabolism. The T-DNA (hog1-5) or 85
transposon (hog1-4) insertion mutants are zygotic embryo lethal, whereas its weak 86
mutation can cause delayed germination, poor growth, reduced seed viability, and 87
reduced whole-genome DNA and histone methylation (Rocha et al., 2005; Mull et al., 88
2006; Baubec et al., 2010; Ouyang et al., 2012). A mutation of folylpolyglutamate 89
synthetase 1 (FPGS1) that converts 5-CH3-THF-Glu1 to 5-CH3-THF-Glun in 90
Arabidopsis can slow germination and reduce levels of whole-genome DNA 91
methylation and H3K9me2 (Zhou et al., 2013). Treatment with sulfamethazine (SMZ), 92
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
5
which is a structural analog and competitor of p-aminobenzoic acid (PABA), the 93
precursor of folate, causes the release of endogenous transposons and repeat elements 94
and the reduction of DNA methylation levels and H3K9me2 (Zhang et al., 2012). A 95
mutation in the cytoplasmic bifunctional methylenetetrahydrofolate 96
dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1) leads to 97
decreased levels of oxidized tetrahydrofolates, DNA hypomethylation, loss of 98
H3K9me and transposon reactivation (Groth et al., 2016). 99
The Arabidopsis genome has 4 MAT genes with different nomenclatures 100
(MAT1/SAM1/AT1G02500, MAT2/SAM2/AT4G01850, MAT3/AT2G36880, 101
MAT4/MTO3/SAMS3/AT3G17390) in different publications (Peleman et al., 1989; 102
Peleman et al., 1989; Goto et al., 2002; Mao et al., 2015; Chen et al., 2016). A 103
previous study indicates that SAMS RNAi transgenic rice (Oryza sativa) lines with 104
down-regulation of OsSAMS1, 2 and 3 show reduced histone H3K4me3 and DNA 105
methylation (Li et al., 2011). In Arabidopsis, the pollen expressed MAT3 is required 106
for maintaining histone and tRNA methylation in pollen, and pollen germination and 107
pollen tube growth (Chen et al., 2016). However, the biological roles of other MAT 108
proteins in Arabidopsis epigenetic regulation are still unknown. 109
In this study, we screened a mutagenized population from the transgenic line 110
L119, which harbors two silenced transgenes, Pro35S::NPTII (NEOMYCIN 111
PHOSPHOTRANSFERASE II) and ProRD29A (RESPONSE TO DESSICATION 29A, 112
a stress-inducible promoter)::LUC, and identified the mat4/sams3/mto3 (methionine 113
over-accumulation) mutant (Shen et al., 2002; Jin et al., 2017) that releases the 114
silencing of both genes. We found that the mat4 mutant, harboring a missense point 115
mutation, dramatically decreases SAM content and CHG and CHH methylation and 116
H3K9me2, leading to the activation of some transposable elements. Exogenous 117
additions of SAM to the medium partially restored histone methylation levels in mat4. 118
The mat1, 2 or 3 mutants reduced SAM content and DNA methylation to a lesser 119
extent than did mat4, indicating a predominant role of MAT4 among MATs. MAT3 120
showed the highest activity among the four MAT proteins in an in vitro assay. The 121
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
6
expression of MAT4 in seedlings was much higher than MAT1, MAT2 and MAT3. 122
The MAT4 promoter driving MAT1, MAT2 or MAT3 cDNA could complement the 123
mat4 mutant, while most transgenic lines carrying the MAT1, MAT2, or MAT3 124
promoters driving MAT4 cDNA could not complement the mat4 mutant. The MAT4 125
loss-of-function mutation generated using the CRISPR/Cas9 technique was lethal. We 126
also found that the MAT proteins in Arabidopsis interacted with each other and 127
themselves both in vitro and in vivo, indicating that they may form homologous or 128
heterogeneous oligomers in Arabidopsis. 129
130
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
7
RESULTS 131
Identification and characterization of mat4 132
To further study the mechanisms regulating transcriptional gene silencing (TGS), we 133
obtained the transgenic line L119 carrying ProRD29A::LUC and Pro35S::NPTII in 134
the Columbia gl1 background. In L119, the transgene loci consist of at least two 135
T-DNA insertions, each with two repeats (Supplemental Fig. 1A-F). ProRD29A is a 136
stress inducible promoter that is induced by ABA, low temperatures and high NaCl 137
concentrations (Yamaguchishinozaki and Shinozaki, 1994). The L119 plants were 138
very sensitive to kanamycin (Kan) and showed little luciferase activity; they grew 139
poorly on media containing 25 mg/L Kan (Fig. 2A) and did not emit any fluorescence 140
after NaCl treatment (Fig. 2D), indicating that both ProRD29A::LUC and 141
Pro35S::NPTII are silenced in L119. However, after introducing the defective in 142
meristem silencing 3 (dms3-1) mutation in the RdDM pathway (Kanno et al., 2008) 143
into L119, ProRD29A::LUC, but not Pro35S::NPTII, was reactivated (Supplemental 144
Fig. 1G-J), suggesting that similar to the C24/RD29A::LUC line (He et al., 2009), 145
ProRD29A::LUC is regulated by the RdDM pathway while Pro35S::NPTII is not. 146
The transgenic line L119 was mutagenized by ethyl-methanesulfonate, and the F2 147
population was screened for Kan-resistant mutants. A mutant, named mat4-3, was 148
isolated in this screen (hereafter referred to as mat4) (Fig. 2A). mat4 seeds germinated 149
later (Fig. 2A) and the seedlings were smaller compared with L119, although these 150
seedlings had relatively normal fertility (Supplemental Fig. S2A, S2B). Two alleles of 151
ddm1, ddm1-18 and ddm1-19, were also identified in this system. ddm1-18 [a G-to-A 152
change at position 2803 (counting from the first putative ATG in the coding frame), 153
which causes a stop codon with a TGG to TGA transition; hereafter referred to as 154
ddm1] and ddm1-19 (a G-to-A change at position 3125, which causes a stop codon 155
with a TGG to TGA transition) exhibited more resistance to kanamycin than mat4, 156
whereas L119 seedlings did not survive on the medium containing 25 mg/L Kan 157
(Supplemental Fig. S2C). DDM1 is a nucleosome-remodeling protein involved in 158
facilitating DNA methyltransferase access to heterochromatin to silence certain 159
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
8 transposable elements and repeats in cooperation with the RdDM pathway (Singer et 160
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
9
al., 2001; Zemach et al., 2013). Here, we used ddm1 as a positive control for reduced 161
DNA methylation. Greater increases of NPTII expression and its protein levels were 162
observed in mat4 than in L119, although the NPTII expression and protein levels were 163
less in mat4 than in ddm1 (Fig. 2B, 2C). Treatment with 300 mM NaCl reactivated the 164
expression of ProRD29A::LUC in mat4 (Fig. 2D). Two silenced transgenic loci were 165
reactivated in mat4, which was also observed in ddm1; thus, we detected 166
endogenously silenced genes, DNA repeats and transposable elements. SUPPRESSOR 167
OF DRM1 DRM2 CMT3 (SDC) is regulated by non-CG DNA methylation 168
(Henderson and Jacobsen, 2008); TSIs are endogenous transcriptionally silent 169
information sites regulated by the DNA replication and repair pathway and DNA 170
methylation independent of the RdDM pathway (Andrea Steimer, 2000; Xia et al., 171
2006); AtGP1 is a LTR-Gypsy transposon modulated by the RdDM pathway (He et al., 172
2009; He et al., 2009); 180-bp CEN are centromeric satellite repeats regulated by 173
DNA methylation independent of the RdDM pathway but not by the DNA replication 174
and repair pathway (Bruce P. May, 2005; Xia et al., 2006). The transcript levels of all 175
these loci were higher in mat4 than in L119 but lower than in ddm1 (Fig. 2C). 176
We then cloned the MAT4 gene by map-based cloning. We first crossed the mat4 177
mutant with the wild-type Ler. The 519 F2 plants that were Kan resistant were 178
isolated and used for mapping. We narrowed mat4 to a region between bacterial 179
artificial chromosome (BAC) clones K14A17 and MPK6 on chromosome 3. We 180
sequenced candidate genes in this region and observed that a G-to-A mutation in 181
AT3G17390 changed 246D to 246N (Fig. 2E, Supplemental Fig. 2D). This mutation 182
occurs in a conserved amino acid that is involved in binding methionine during the 183
reaction, according to the crystal structure of human (Homo sapiens) MAT2A, which 184
is different from the mto3 mutation in the ATP binding site (Shen et al., 2002). This 185
point mutation did not cause alteration in the transcript level (Supplemental Fig. S2E). 186
To confirm whether the mutation in MAT4 is responsible for the Kan resistance of 187
mat4, we transformed the full genomic length of MAT4, including the 2221-bp 188
promoter and the full genomic sequence fused with FLAG or a GFP tag into the mat4 189
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
10
mutant. A number of different transgenic lines were obtained and shown to be Kan 190
sensitive, which was also observed in L119. We selected one MAT4-FLAG line (line 1) 191
for further study (hereafter referred as MAT4-FLAG). Immunoblotting using 192
anti-FLAG antibodies indicated that MAT4-FLAG expressed the MAT4-FLAG protein 193
(Supplemental Fig. S2F). MAT4-FLAG was sensitive to Kan and had normal 194
germination (Fig. 2F), and the NPTII protein level was restored to the basal level 195
observed in L119 (Fig. 2G), suggesting that AT3G17390 could complement the mat4 196
mutant phenotype. Reverse transcription quantitative PCR (RT-qPCR) analyses 197
indicated that the expression of NPTII, and certain endogenous loci in the 198
MAT4-FLAG also returned to the basal level observed in L119 (Supplemental Fig. 199
S2G). We did not observe any enhanced severe phenotypes of the mat4 mutant after 200
several generations. We used the egg cell-specific CRISPR/Cas9 system in L119 201
(Wang et al., 2015) and created two MAT4 knock-out lines mat4-c19 and mat4-c32. 202
mat4-c19 has a fragment deletion from 76 to 180 bp (counting from the first putative 203
ATG) and mat4-c32 from 706 to 797 bp (Supplemental Fig. S2H). However, we were 204
unable to obtain homozygous mat4 mutants, indicating that MAT4 is an essential gene 205
in Arabidopsis. 206
The subcellular localization in transgenic L119 plants expressing 207
Pro35S::MAT4-GFP indicated that MAT4-GFP was localized in the nucleus and 208
cytosol (Fig. 2H a). A similar localization of MAT4-GFP was observed in a transient 209
transformation assay using Arabidopsis protoplasts and Nicotiana benthamiana leaves 210
(Fig. 2H b, c). To avoid the possibility that GFP translocates to the nucleus by itself, 211
we isolated the cytosol and nuclei from L119 and the MAT4-FLAG transgenic line and 212
performed an immunoblot assay. MAT4-FLAG protein was detected in both the 213
cytosol and the nucleus (Fig. 2I). Previous studies also indicate that SAM1/MAT1, 214
SAM2/MAT2 and MAT3 are all localized in both cytosol and nuclei (Mao et al., 2015; 215
Chen et al., 2016). These results suggest that MAT proteins may function in both the 216
cytosol and the nucleus in Arabidopsis. 217
DNA methylation of transgenic loci is reduced in mat4 218
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
11
MAT4 catalyzes the biosynthesis of SAM, which is a universal methyl group donor 219
for DNA and histone methylation; thus, mat4 may reactivate the silenced 220
Pro35S::NPTII and ProRD29A::LUC because of decreased DNA and/or histone 221
methylation. Bisulfite sequencing analyses indicated that CHG and CHH methylation 222
of transgenic and endogenous RD29A promoters largely decreased in mat4 compared 223
with that in L119 (Fig. 3B, 3C), which was consistent with the data from 224
whole-genome bisulfite sequencing (Fig. 3D). However, CHG and CHH methylation 225
decreased to a lesser extent at the 35S promoter in mat4 compared with that in L119 226
(Fig. 3A, 3D). However, only limited changes were observed in the CG methylation 227
with transgenic RD29A and 35S promoters in mat4. In contrast, ddm1 greatly reduced 228
CG methylation and only moderately affected CHG and CHH methylation, except for 229
the transgene RD29A promoter, in which ddm1 did not affect CHH methylation. In 230
addition, the DNA methylation of the MAT4-FLAG line was consistently restored to 231
the L119 level (Fig. 3A, 3B, 3C). These results indicate that mat4 reduces DNA 232
methylation in transgenes in L119. 233
mat4 reduces DNA methylation at the whole-genome level 234
We compared the DNA methylation level of mat4 with that of L119 at the 235
whole-genome level by bisulfite sequencing. We obtained nearly 5G raw data 236
including adapter and low-quality data for each sample, from which we obtained 4.2G 237
clean data for our subsequent analyses. The total reads were mapped to the genome of 238
TAIR 10. We then obtained the methylation level of CG, CHG, and CHH by 239
calculating the ratio of C to C+C/T using the tool of Bismark (Krueger and Andrews, 240
2011) (Fig. 4A). We also included the previously published data of ddm1 (Zemach et 241
al., 2013) for comparison. The methylation levels of CG (22.9%), CHG (4.2%), and 242
CHH (1.4%) in mat4 were lower than the levels of CG (25.2%), CHG (8.2%), and 243
CHH (2.4%) in L119 (Fig. 4A). CHG and CHH methylation in mat4 decreased by 244
nearly half, whereas CG methylation decreased approximately by 9.2%, suggesting 245
that mat4 has different effects on CG, CHG and CHH DNA methylation (Fig. 4B). 246
mat4 displayed a relatively smaller reduction of CG and CHG methylation and greater 247
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
12
reduction of CHH methylation compared with that observed in ddm1 (Fig. 4B). 248
Frequency distribution histograms of significant methylation differences between 249
L119 and mat4 in CG, CHG, and CHH also indicated that the CHG and CHH 250
methylation dramatically decreased in mat4 (Fig. 4C). 251
To determine the distribution of changes in the DNA methylation patterns in 252
detail, we calculated the DNA methylation 2 kb upstream and downstream of the 253
genes and transposable elements (TEs), respectively. In genes that exclude TEs or 254
repeats, CG methylation mainly occurred in the gene bodies. mat4 reduced the CG 255
methylation in the gene body regions by approximately 2.5% (Fig. 4D). However, 256
ddm1 showed greater reductions in CG methylation compared with mat4 in these 257
regions (Fig. 4D). CHG and CHH methylation did not show noticeable changes (Fig. 258
4D) because these regions exhibit limited CHG and CHH methylation. For TEs, we 259
focused on two TE types: TEs shorter than 0.5 kb (S-TEs, usually regulated by the 260
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
13
RdDM pathway) and TEs longer than 4 kb (L-TEs, usually regulated by the DDM1 261
pathway) (Teixeira et al., 2009; Zemach et al., 2013). Generally, mat4 displayed less 262
CG, CHG and CHH methylation than the L119 in both S-TEs and L-TEs (Fig. 4E, 4F). 263
Compared with ddm1, mat4 displayed more CG methylation in both short and long 264
TEs and more CHG methylation in long TEs. However, mat4 showed similar CHG 265
methylation in short TEs and CHH methylation in long TEs as ddm1 (Fig. 4E, 4F). 266
mat4 displayed less CHH methylation in short TEs than ddm1, which is consistent 267
with previous studies of DDM1 regulation of DNA methylation in long TEs, but not 268
short TEs that are mostly regulated by the RdDM pathway (Teixeira et al., 2009; 269
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
14
Zemach et al., 2013). These results suggest that mat4 mainly reduces CHG and CHH 270
methylation and, to a lesser extent, CG methylation. When mapping these 271
hypo-differentially methylated regions (hypo-DMRs) to the five chromosomes, we 272
found that the distribution of these hypo-DMRs were concentrated around the five 273
centromeres, which displayed a dramatic decrease of CHG and CHH methylation 274
(Supplemental Fig. S3, Supplemental data set S1-S3). These results indicate that mat4 275
reduces genomic-wide DNA methylation, especially CHG and CHH methylation at 276
pericentromeric heterochromatin regions. 277
mat4 decreases histone modifications in heterochromatin regions 278
Because DNA methylation, especially CHG and CHH methylation, is reduced 279
throughout the genome in mat4, we sought to determine whether mat4 has an effect 280
on histone methylation. We verified the histone modifications in H3K9me2, 281
H3K9me1, and H3K27me1 because these modifications usually accompany DNA 282
methylation in heterochromatin regions, and we also verified the modifications in 283
H3K4me3 because this modification accompanies high gene expression (Tariq et al., 284
2003; Jacob et al., 2009). Using immunoblotting assays, we found that the H3K9me2 285
levels in mat4 were comparable to those in ddm1, and greatly reduced compared with 286
that of L119. In addition, only a small decrease in H3K9me1 was exhibited in mat4, 287
whereas a dramatic decrease was observed in ddm1 compared with L119 (Fig. 5A, 288
5B). Both mat4 and ddm1 had a lower H3K27me1 level than L119. In the mat4 289
complementary line, both H3K9me2, H3K9me1 and H3K27me1 were restored to the 290
wild-type level, whereas in mat4, ddm1, L119 or MAT4-FLAG, H3K4me3 was not 291
changed (Supplemental Fig. S4A, S4B). 292
We then compared the heterochromatin status in nuclei using an 293
immunofluorescence assay with different antibodies. In the wild-type cells, more than 294
89% of the interphase nuclei showed H3K9me2, H3K9me1 and H3K27me1 295
immunofluorescence associated with the condensed pericentromeric heterochromatin 296
regions stained with 4',6-diamidino-2-phenylindole (DAPI). However, approximately 297
78% of mat4 and 87% of ddm1 nuclei showed chromocenter decondensation and 298
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
15
reduced H3K9me2 immunofluorescence. ddm1 showed strongly reduced H3K9me1 299
immunofluorescence, which was not observed in mat4, whereas ddm1 and mat4 300
mutants showed substantially reduced H3K27me1 immunofluorescence (Fig. 5C-E). 301
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
16
In MAT4-FLAG, H3K9me2 immunofluorescence was restored to the L119 level 302
(Supplemental Fig. S4C, S4D). However, we did not detect a difference in H3K4me3 303
immunofluorescence in L119, mat4 and MAT4-FLAG (Supplemental Fig. 4E, 4F). We 304
confirmed the decrease of H3K9me2 at certain loci using chromatin 305
immunoprecipitation (ChIP)-PCR (Fig. 5F). These results suggest that mat4 reduces 306
the histone methylation in heterochromatin regions, especially H3K9me2 and 307
H3K27me1. 308
mat4 reactivates silenced TEs 309
To determine how mat4 modulates gene expression, we performed RNA sequencing 310
(RNA-seq). Total RNA was extracted from 15-day-old seedlings and then subjected to 311
RNA-Seq with two biological replicates. We obtained 3G clean data with each 312
replicate, mapped all of the obtained reads to TAIR 10, and then compared the 313
transcript levels between mat4 and L119 using edgeR (Robinson et al., 2010). We 314
obtained 1284 protein coding genes and 364 TEs with transcript level changes of at 315
least two-fold and P<0.0001. Among these protein-coding genes, 66% (842) were 316
up-regulated and 34% (442) were down-regulated (Fig. 6A, 6B, Supplemental data set 317
S4-7). After mapping these genes on the five chromosomes, we found that they were 318
evenly distributed along the chromosomes arms and rarely localized at the centromere 319
regions (Fig. 6C). Approximately 92% (334) of the differentially-expressed TEs were 320
up-regulated and concentrated around the centromeres (Fig. 6C). The expression of 321
certain genes and TEs was confirmed by RT-qPCR in L119, mat4 and MAT4-FLAG 322
(Supplemental Fig. S5A, S5B). Compared with previously published data in ddm1 323
and fpgs1, we found that 127 up-regulated TEs in mat4 were also up-regulated in 324
ddm1 and fpgs1 with reduced DNA methylation and H3K9me2 (Fig. 6D) (Zemach et 325
al., 2013; Zhou et al., 2013). After dividing the up-regulated TEs according to their 326
characteristics, two categories of TEs, long terminal repeat (LTR) /Gypsy and 327
Enhancer/Suppressor Mutator (En/Spm)-like transposons (Fig. 6E), accounted for 328
nearly 50% of all of the up-regulated TEs in mat4. Alterations to DNA methylation 329
were not associated with the expression of protein coding genes, however, reduced 330
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
17
DNA methylation in mat4 was closely associated with increased TE expression 331
(Supplemental Fig. S5C). When comparing the CHG hypo-DMRs and up-regulated 332
TEs in mat4 with the published data in suvh4/5/6 and cmt3 (Stroud et al., 2013), we 333
found that these mutants had higher overlap than that by chance as viewed in VENNY 334
diagram (Fig. S5D), indicating that MAT4 affects a large number of targets shared 335
with those methyltransferases. In conclusion, mat4 led to the activation of the silenced 336
transposons as a result of the reduction in DNA methylation and histone methylation. 337
Application of SAM partially rescues the phenotype of mat4 338
To test whether the decreases of DNA and histone methylation were caused by the 339
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
18
alteration of SAM content in mat4, we measured SAM contents in mat4, L119 and 340
MAT4-FLAG by liquid chromatography-mass spectrometry (LC-MS). The SAM 341
content in mat4 was decreased by nearly 35% compared to that in L119 (Fig. 7A). 342
Interestingly, the content of SAH, which is a strong inhibitor of SAM-dependent 343
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
19
methyltransferases, was significantly increased in mat4, leading to a large decrease in 344
the ratio of SAM/SAH, which is an important index influencing the methylation status 345
(Fig. 7B). Meanwhile, the contents of SAM and SAH in MAT4-FLAG were restored 346
to L119 level (Fig. 7A, 7B). Therefore, we sought to determine whether the exogenous 347
application of SAM could rescue the phenotype of mat4. After adding 400 mg/L SAM 348
to the medium, the Kan susceptibility of mat4 was partially rescued (Fig. 7C, 7D). An 349
analysis of the transcript levels of transgenic and endogenous genes indicated that the 350
application of SAM inhibited the high expression of these genes in mat4 (Fig. 7E). An 351
immunofluorescence assay indicated that H3K9me2 levels were restored by the 352
application of SAM (Fig. 7F, 7G). These results suggest that the decreased SAM in 353
mat4 leads to the release of the silencing of these tested genes. 354
MAT4 plays a predominant role in SAM production and DNA methylation 355
among different MAT homologs 356
In Arabidopsis, four MAT homologs present near 90% identity between each other in 357
their amino acid sequences (Shen et al., 2002; Lindermayr et al., 2006). Because mat4 358
reduces SAM content and DNA methylation, we sought to determine whether other 359
MAT mutants have similar roles. We obtained three T-DNA lines: SALK_059210 360
carrying a T-DNA insertion in the C-terminus of AT1G02500 (mat1); SALK_052006 361
carrying a T-DNA insertion in the N-terminus of AT4G01850 (mat2), and 362
SALK_019375 carrying a T-DNA insertion after the putative stop codon of 363
AT2G36880 (mat3), which was used in the previous study (Chen et al., 2016). All 364
three T-DNA insertion lines greatly reduced the expression of each targeted gene 365
(Supplemental Fig. S6A). We measured the contents of SAM in these mutants, and 366
found that SAM content in mat1 and mat2 decreased only about 6% compared to that 367
in L119, but no clear change was observed in mat3 (Supplementary Fig. S6B), which 368
is consistent with its main expression in pollen (Chen et al., 2016). We further 369
measured the DNA methylation level in these mutants at the whole genomic level by 370
bisulfite sequencing, and found that the DNA methylation in CG, CHG and CHH 371
slightly decreased in these mutants (Supplementary Fig. S7A), among which mat3 372
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
20
CHG and CHH methylation was reduced to a lesser extent than mat1 or 2 in Long and 373
Short-TEs (Supplementary Fig. S7B, S7C), which was consistent with the SAM 374
contents in these mutants. These results indicate that MAT1, 2 and 3 have less of an 375
effect on DNA methylation than MAT4 in seedlings. However, MAT3 may play a 376
major role in pollens (Chen et al., 2016). 377
In order to clarify the effect of these MAT proteins on expression of 378
Pro35S::NPTII in L119, we created loss of function mutants of MAT1, 2, 3 genes 379
using the egg cell-specific CRISPR/Cas9 system in the L119 background 380
(Supplementary Fig. S8A) (Wang et al., 2015). mat1-c3 was a single base insertion 381
mutant, in which an A insertion after 421 bp (counting from the first putative ATG), 382
led to a frame-shift mutation; mat1-c19 had a fragment deletion from 222 bp to 450 383
bp; mat2-c6 was a single base deletion mutant at 488 bp, leading to a frame-shift 384
mutation; mat2-c13 had a fragment deletion from 490 bp to 569 bp; mat3-c8 had a 385
fragment deletion from 366 bp to 420 bp. The expression of each target gene was 386
greatly reduced compared to the wild type (Supplemental Figure S8A). All these 387
mutants were sensitive to Kan (Supplementary Fig. S8B) and the transcriptional levels 388
of NPTII did not differ with that in L119 (Supplementary Fig. S8C), indicating that 389
the mutations in MAT 1, 2 and 3 do not release the silencing of Pro35S::NPTII, which 390
was consistent with the results of a smaller reduction in DNA methylation in their 391
T-DNA lines. The mat3-c8 mutant produced 1–2 seeds per silique, which is similar 392
with previous results (Chen et al., 2016). 393
The expression pattern of MAT4 determines its predominant biological roles in 394
Arabidopsis 395
Although the amino acid sequences of the four MATs shared a high percentage of 396
identity, these proteins did not compensate for each other in plants. Whether their 397
expression patterns or protein activities determined their specificity is not known. To 398
address this question, firstly, we compared the catalytic activities of those four 399
proteins in vitro. The MAT proteins were expressed in and purified from Escherichia 400
coli. MAT1, MAT2 and MAT4 exhibited similar activity, while MAT3 had higher 401
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
21
activity than other three (Fig. 8A). The amount of SAM produced increased with 402
increasing MAT4 protein concentration as well (Fig. 8B). However, we could not 403
detect any activity for the MAT4D246N mutant protein (Fig. 8B), indicating that the 404
mutant protein largely loses its activity in vitro. 405
We used the MAT4 promoter driving MAT1, MAT2 or MAT3 cDNA to evaluate 406
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
22
whether these cDNAs could complement mat4. Here we fused MAT cDNA with GFP 407
to observe its expression. We obtained 12 mat4 transgenic lines carrying 408
ProMAT4::MAT1-GFP, 8 carrying ProMAT4::MAT2-GFP, and 19 carrying 409
ProMAT4::MAT3-GFP. These transgenic plants had high GFP fluorescence. All these 410
transgenic plants had high expression of transgenes and complemented mat4 mutant 411
phenotypes (Fig. 8C-E). These results indicated that MAT proteins have comparable 412
biological functions in plants. 413
A previous study indicates that transforming Pro35S::MAT2 into mto3-1 (the 414
mat4 allele) failed to complement the mto3 phenotype (Shen et al., 2002). Given that 415
MAT1 and MAT2 are expressed in most plant tissues (Peleman et al., 1989; Mao et al., 416
2015) and MAT3 is mainly expressed in pollens (Chen et al., 2016), we used MAT1, 2, 417
or 3 promoters driving MAT4 cDNA to examine whether they could complement mat4. 418
We obtained 27 mat4 transgenic lines carrying ProMAT1::MAT4-GFP, 5 carrying 419
ProMAT2::MAT4-GFP and 6 carrying ProMAT3::MAT4-GFP. We found that all 420
ProMAT2::MAT4-GFP or ProMAT3::MAT4-GFP and most ProMAT1::MAT4-GFP 421
transgenic plants did not complement the mat4 Kan resistant phenotype because they 422
had lower GFP levels, as indicated by fluorescence imaging and immunoblotting 423
using GFP antibodies (Figure 9A-D). In contrast, ProMAT4::MAT2-GFP transgenic 424
lines had higher GFP levels (Figure 9A-D). In 27 ProMAT1::MAT4-GFP transgenic 425
lines, 7 lines showed different Kan sensitive phenotypes. We selected three lines and 426
compared their Kan sensitivity with other lines. These three lines showed more Kan 427
sensitivity than other lines (Supplemental Fig. 9A), indicating that they complemented 428
or partially complemented the mat4 mutant. GFP protein levels were higher in these 429
complemented lines than in non-complemented lines as indicated by GFP 430
fluorescence and protein immunoblotting (Supplementary Fig. S9B-D). The 431
expression levels of transgenes were also higher in these complemented lines than 432
others (Supplementary Fig. S9E). The higher expression of ProMAT1::MAT4-GFP 433
may be caused by the different genomic site in which the T-DNA was inserted. These 434
results indicate that the expression level of MAT genes determined their biological 435
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
23
roles in Arabidopsis. 436
We performed further genetic analyses among these mutants. mat1mat2 double 437
mutants did not show any growth or developmental differences from the wild-type 438
plants (Supplementary Fig. S10A). mat1-c19 mat4 double mutant seedlings were 439
much smaller than the wild type, and did not produce any seeds (Fig. 9E). We could 440
not obtain mat2-c13 mat4 homozygous double mutants because the double mutants 441
had embryonic defects (Fig. 9F, 9G, Supplementary Fig. S10B). These results indicate 442
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
24
that the expression pattern of MAT4 gene determines its predominant biological roles 443
in Arabidopsis. 444
MATs form homologous or heterologous oligomers in cells 445
To explore the functions of MAT4, we tried to identify the MAT4-interacting proteins 446
by immunoprecipitation followed by mass spectrometry analysis using the 447
complementary transgenic line MAT4-FLAG. We precipitated MAT4-FLAG with 448
anti-FLAG beads, and used the L119 lines as negative controls. We identified MAT1, 449
MAT2 and MAT3, each with unique peptides from MAT4-FLAG 450
co-immunoprecipitation (co-IP) proteins (Supplemental table S1). Then, we 451
confirmed their interactions in vivo using co-immunoprecipitation assays in 452
Arabidopsis protoplasts transiently expressing different proteins (Fig. 10A). In E. coli, 453
when co-expressing MAT4-His with GST-MAT1, GST-MAT2, GST-MAT3, 454
GST-MAT4 or only GST (as the negative control), respectively, we found that each of 455
them, but not GST, could be purified together with MAT4-His (Fig. 10B), suggesting 456
that MAT4 can interact with MAT1, MAT2, MAT3 and itself in vitro. We further 457
confirmed that MAT1, MAT2 and MAT3 were able to interact with each other and 458
themselves in both in vivo and in vitro assays (Supplementary Fig. S11, S12). Next, 459
we carried out gel filtration using the proteins isolated from the MAT4-FLAG 460
transgenic complementary line. We observed three peaks from the eluted fractions 461
(Fig. 10C). LC-MS analyses of each peak authenticated four MAT proteins (Fig. 10D), 462
indicating that these MATs can form different sizes of homologous or heterologous 463
oligomer complexes in vivo, which merits further examination. 464
465
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
25
DISCUSSION 466
SAM provides methyl groups for numerous methyltransferases in transmethylation 467
reactions, including DNA and histone methylations in all living cells. In this study, we 468
identified MAT4 because its mutation reactivates the silenced Pro35S::NPTII and 469
ProRD29A::LUC in L119. MAT is well conserved during evolution, and it usually has 470
three domains: the N-terminal domain, the C-terminal domain and the central 471
M-domain (Fusao Takusagawa, 1996). In Arabidopsis, there are four homologs of 472
MAT, MAT1-4; these homologs share nearly 90% amino acid sequence identity 473
(Peleman et al., 1989; Peleman et al., 1989; Shen et al., 2002). mto3, an allele of mat4, 474
was isolated in a screen based on ethionine (a toxic analog of methionine) sensitivity. 475
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
26
The level of methionine is increased more than 200-fold and the concentration of 476
SAM is decreased by 35% compared with the wide type (Shen et al., 2002). In this 477
study, we found that both DNA and histone methylation were largely reduced as a 478
result of the decrease of SAM content in mat4. Our study provides direct evidence for 479
the importance of SAM in providing methyl donors and modulating epigenetic status. 480
In theory, SAM is a general methyl group donor and the reduction of SAM 481
should have an unbiased effect on DNA and histone methylations. However, we 482
found that the reduction of DNA and histone methylation was uneven, with mat4 483
showing large decreases in CHG and CHH methylation as well as H3K9me2 and 484
H3K27me1 (Fig. 4, 5). Changes in H3K4me3 were not observed and CG methylation 485
decreased to a lesser extent. In animals, the supplementation of methionine, an 486
essential amino acid, can modulate the SAM/SAH ratio and impact H3K4me3 487
(Mentch et al., 2015). Threonine, another essential amino acid, is the major fuel 488
source for glycine, acetyl-CoA and SAM. Restricted applications of threonine can 489
reduce H3K4me3 levels, which results in slower growth and increased differentiation 490
in mouse embryonic stem cells (Shyh-Chang et al., 2013). In addition, in SAMS RNAi 491
rice, H3K4me3 is significantly reduced (Li et al., 2011). These results suggest that 492
SAM limitation can result in different changes in DNA and histone modifications in 493
different species. These differences can be explained by several factors. First, 494
different methyltransferases might have different SAM concentration thresholds. In 495
addition, MET1 and H3K4 methyltransferases might efficiently use low 496
concentrations of SAM to complete the reactions in mat4, whereas the histone 497
methyltransferases for H3K9me2 and DNA methyltransferases for CHG and CHH 498
might have lower activity at such concentrations. Second, the reinforcing loop 499
between CHG methylation and H3K9me2 cannot be maintained and is even disrupted 500
in mat4, which would lead to a serious reduction in methylation for both. Third, the 501
increased SAH in mat4 would compete with SAM and decrease SAM accessibility to 502
methyltransferases, which mostly reduced the CHG and CHH methylation and 503
H3K9me. Similar results have been observed in both fpgs1 and mthfd1 mutants (Zhou 504
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
27
et al., 2013; Groth et al., 2016). Both fpgs1 and mthfd1 mutants accumulate relatively 505
more SAH, which leads to a decreased ratio of SAM/SAH (Zhou et al., 2013; Groth et 506
al., 2016). SAH is a strong inhibitor that competes with SAM for SAM-dependent 507
transmethylation (De La Haba and Cantoni, 1959). These studies suggest that the 508
CMT3 or the CMT2 pathway has a positive feedback circuit with SUVH4 509
(KRYPTONITE)/5/6 to maintain CHG or CHH methylation and H3K9me (Zhou et al., 510
2013; Stroud et al., 2014; Groth et al., 2016). Consistent with the reduced DNA 511
methylation, our RNA-seq data indicated that a large number of TEs were activated in 512
the mat4 mutant. These up-regulated TEs were enriched around the heterochromatin 513
regions of the centromeres, which are largely shared with those found in ddm1 and 514
fpgs1 mutants. Given that reduced DNA methylation is found only at certain sequence 515
contexts, it is also possible that the inefficient histone methylation might indirectly 516
affect DNA methylation. For example, the CHG DNA methylation in the 517
Pro35S-NPTII transgene was only moderately reduced, while a more significant 518
reduction in H3K9me2 was found in the 35S promoter of the mat4 mutant. However, 519
this hypothesis is hard to test as SAM is a common substrate for both DNA and 520
histone methylation. Reduced SAM must more or less affect both DNA and histone 521
methylation. 522
In humans, three MAT genes encode MATα1, MATα2 and MATβ. MATα1 and 523
MATα2 can form homo- dimers or tetramers that have different affinity for substrates, 524
and MATα2 can interact with MATβ to strengthen the activity of (MATβ)4 (Murray et 525
al., 2014). In Saccharomyces cerevisiae, when two MATs that share 92% identity in 526
amino acid sequence are disrupted, the mutants display opposite phenotypes to the 527
excess ethionine added in the growth medium (Thomas and Surdin-Kerjan, 1987; 528
Thomas et al., 1988; Thomas and Surdin-Kerjan, 1991), indicating that different MAT 529
isoforms act on their own rhythms. There are four close MAT homologs in 530
Arabidopsis. However, we found that in the in vitro assays MAT3 has the highest 531
activity, while MAT1, MAT2 and MAT4 have comparable but lower activities. Among 532
them, MAT4 is predominant as its missense mutation reduces SAM and DNA 533
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
28
methylation to greater extent than MAT1 and MAT2 loss-of-function mutations, and 534
can release the silencing of Pro35S::NPTII, while other mutations cannot. We used 535
the CRISPR/Cas9 technique, but failed to get the loss-of-function homozygous 536
mutant of MAT4. These results indicate that MAT4 is an essential gene for plant 537
growth and development in Arabidopsis. We found that the MAT4 promoter driving 538
different MAT cDNAs can complement mat4 mutants. However, only a few mat4 539
mutants can be complemented by the MAT1 promoter driving MAT4 cDNA, which 540
might be caused by high expression of ProMAT1::MAT4-GFP in these transgenic 541
lines, likely because the T-DNAs were inserted in environment-friendly sites in the 542
genomic region. Nevertheless, mat4 mutants were not complemented in several 543
ProMAT2::MAT4-GFP and ProMAT3::MAT4-GFP transgenic lines. These results 544
indicate the expression pattern of MAT4, but not MAT4 protein itself, is important for 545
its predominant biological roles in Arabidopsis. Using pull-down assays and co-IP 546
assays, we found that MATs interacted with each other both in vitro and in vivo, 547
suggesting that MATs can also form homo-, and/or hetero- oligomers of different sizes 548
in Arabidopsis. However, more attempts or even crystallographic structural analyses 549
should be carried out to obtain more information about their precise composition in 550
Arabidopsis. 551
Materials and Methods 552
Plant Growth Conditions, Mutant Screening and Identification 553
Arabidopsis (Arabidopsis thaliana) seeds were sterilized with 0.5% NaClO and then 554
sown into Murashige and Skoog (MS) medium, which contained 2% (weight/volume) 555
sucrose and 0.8% (w/v) agar. After 3 days at 4°C, the plates were transferred to 556
growth chambers with long-day conditions (23 h of light / 1 h of dark) at 22°C. 557
Generally, 10-day seedlings were transferred to soil and cultured in a greenhouse with 558
long-day conditions (16 h of light / 8 h of dark) at 20°C. 559
We used L119, which harbors two transgenes, ProRD29A::LUC and 560
Pro35S::NPTII, as the wild-type line. The mutants were selected from an 561
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
29
ethyl-methanesulfonate mutagenized population of L119 for resistance to 25 mg/L 562
Kan, whereas the L119 lines are typically sensitive under this condition. Map-based 563
cloning was conducted to identify the mutation. We crossed our mutants with Ler and 564
obtained the F2 population, from which we selected Kan-resistant lines (519 total) for 565
mapping. 566
For mutant complementation, the full genomic length of MAT4, including the 2221 bp 567
promoter and the overall genomic sequence, was cloned into pCAMBIA1307. The 568
construct in Agrobacterium tumefaciens strain GV3101 was transformed into mat4 by 569
the floral dip method (Clough and Bent, 1998). The homozygous transgenic lines 570
were selected on MS medium supplemented with 30 mg/L hygromycin from the next 571
T2 generation. All of the primers used in this study are listed in the Supplemental 572
Table S2. 573
RNA Analysis 574
For real-time reverse transcription quantitative PCR (RT-qPCR), total RNA was 575
isolated using TRIzol reagent (Invitrogen) from 15-day seedlings, and 4 µg RNA was 576
reverse-transcribed into cDNA using the GOSCRIPT reverse transcription system 577
(Promega A5001). Then, 2 µL of diluted (10X) cDNA mixture was used as the 578
template for a PCR assay using 20 µL of SYBR Green Master Mix (TaKaRa) 579
performed on a Step One Plus system (Applied Biosystems). The experiments were 580
performed in three independent biological replicates with technical triplicate. All of 581
the primers used in the real-time PCR assay are listed in Supplemental Table S2. 582
Subcellular Localization 583
The full-length cDNA of MAT4 fused with a GFP tag under the control of the super 584
promoter was constructed in the pCAMBIA 1300 vector and the full genomic length 585
of MAT4, including the 2221 bp promoter and the overall genomic sequence, was 586
cloned into pCAMBIA1300. The plasmids were extracted and purified with the 587
Plasmid Maxprep Kit (VIGOROUS N001). Then, we introduced the plasmids into 588
Arabidopsis protoplasts as previously described (Kong et al., 2015). After 14-16 h of 589
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
30
incubation in light, the protoplasts were viewed using a confocal microscope (Zeiss 590
LSM 510 META), and the GFP signal was detected with 488 nm excitation. Empty 591
GFP plasmids were used as a control. A. tumefaciens strain GV3101 carrying the 592
same constructs were also injected into N. benthamiana leaves. After 48-72 hours of 593
incubation, a section of the injected leaves was examined using the confocal 594
microscope, and the GFP signal was detected using 488 nm excitation. An empty GFP 595
construct was used as a control. We also obtained transgenic lines by transforming 596
this construct into L119 using A. tumefaciens strain GV3101. The 7-day growth of the 597
T2 homozygous transgenic plants was used to detect fluorescence signals using a 598
confocal microscope (Zeiss LSM 510 META) with 488 nm excitation. 599
Cellular Distribution of MAT4-FLAG 600
Next, 0.1 g of 15-day seedlings was ground into powder in liquid nitrogen and 601
suspended with 200 µL isolation buffer (0.4 M sucrose, 10 mM Tris∙HCl (pH=8.0), 10 602
mM MgCl2, 5 mM β-Mercaptoethanol, 1 mM PMSF), then filtered through 603
microcloth (Calbiochem: 475855-1R), and the flow-through was centrifuged at 2800 g 604
for 10 minutes at 4°C. The supernatant was used for the cytosol, while the precipitate 605
was used for the nuclei after four washes with isolation buffer. Then immunoblotting 606
using antibodies (H3: Millipore, 07-690; FLAG: Sigma-Aldrich, F3165; PEPC: 607
Agrisera, AS09458) was carried out. Here, phosphoenolpyruvate carboxylase (PEPC) 608
was used as the cytosol marker, while H3 was used as the nucleus marker. 609
Bisulfite Sequencing 610
Genomic DNA was extracted using the DNeasy Plant Mini Kit (QIAGEN 69104) 611
from 15-day seedlings. The EZ Methylation-Gold Kit (Zymo Research D5005) was 612
used to analyze DNA methylation. Five hundred nanograms of DNA was added to the 613
reaction, and all steps followed the protocol supplied in the kit. Nearly 50 ng treated 614
DNA was added to the PCR reaction using the specific primers listed in Supplemental 615
Table 2. The PCR products were introduced into the pMD18-T simple vector (Takara 616
6011), and at least 15 clones were sequenced for each sample. 617
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
31
Histone Extraction and Immunoblotting 618
Histone proteins were extracted from 15-day seedlings following the protocol as 619
previously described (Li et al., 2012). The antibodies used in immunoblotting were 620
H3 (Millipore: 07-690), H3K9me1 (Millipore: 17-680), H3K9me2 (Abcam: ab1220), 621
H3K27me1 (Millipore: 07-448), and H3K4me3 (Millipore: 07-473). H3 was used as 622
the loading control. The experiments were performed in three independent biological 623
replicates. 624
Histone Immunofluorescence Staining Assay 625
The assay mainly followed a previously described process (Soppe et al., 2002) with 626
subtle modifications. The nuclei were isolated from 15-day seedlings. After 627
resuspending with sorting buffer, the nuclei were dropped on the slides to air dry. The 628
nuclei were then post-fixed using 4% paraformaldehyde in PBS for 20 minutes, 629
washed four times with PBS, and closed with signal enhancer (Cell Signalling: 11932) 630
for 30 minutes at room temperature. After washing four times, the plates were 631
incubated with primary antibody for 2 hours at 37°C or overnight at 4°C covered with 632
parafilm. Then, the plates were washed four times in vats filled with PBST (PBS 633
added with 0.1% TWEEN20), each for five minutes, and incubated with secondary 634
antibody in dark for 1.5 hours at room temperature. Then, the plates were washed four 635
times in vats filled with PBST. Eight microliters of 4',6-diamidino-2-phenylindole 636
(DAPI) (1 µg/mL) was added onto the slides to counterstain the nuclei. The slides 637
were covered with cover glasses. The signal was observed with a confocal microscope 638
(Leica sp5) and collected under the emission wave-length of 405 nm and 561 nm for 639
DAPI and Rhodamine. 640
The primary antibodies used in this assay were H3K9me1 (Millipore: 17-680, 641
1:50, Rabbit), H3K9me2 (Abcam: ab1220, 1:100, Mouse), H3K27me1 (Millipore: 642
07-448, 1:100, Rabbit), H3K4me3 (Millipore: 07-473, 1:100, Rabbit). Secondary 643
antibodies used in this assay were Rhodamine Red conjugate-Goat anti-mouse 644
(Invitrogen R6393) and Rhodamine Red conjugate-Goat anti-rabbit (Invitrogen 645
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
32
R6394). 646
Whole-genome Bisulfite Sequencing and Analyses 647
Genomic DNA was extracted from 15-day seedlings using DNeasy Plant Mini Kit 648
(QIAGEN 69104). Two micrograms DNA was used for bisulfite treatment and library 649
construction, and MethylC-seq was carried out using HiSeq 2000 (Illumina). 650
Raw data were obtained from the whole-genome bisulfite sequencing using the 651
Illumina HiSeq platform. Clean data were generated by trimming adaptor bases and 652
removing low quality reads. For data analysis, paired-end clean reads were mapped to 653
the reference genome sequence of the Arabidopsis genome (TAIR10) with Bismark 654
(Krueger and Andrews, 2011). The DMRs (differentially-methylated regions) were 655
determined and identified as previously published (Zhao et al., 2014). 656
For investigation of DMR enrichment, we followed the previously described 657
analysis with some modifications (Zhao et al., 2014). The DNA methylation level in 658
genes without transposable elements (TEs), short TEs (shorter than 0.5 kb) and long 659
TEs (longer than 4 kb) were calculated. 660
RNA Sequencing and Analysis 661
Total RNA was extracted from 15-day-old seedlings using the RNeasy Plant Mini Kit 662
(QIAGEN 74904). Two µg RNA was used for library construction, each sample with 663
two replicates. The transcriptome data set used in this study was obtained using the 664
Illumina HiSeq platform, and 125 bp trimmed paired-end reads with high quality were 665
generated. The trimmed reads were mapped to the reference genome sequence of the 666
Arabidopsis genome (TAIR10) using bowtie2 667
(http://computing.bio.cam.ac.uk/local/doc/bowtie2.html) with default settings 668
(Langmead et al., 2009). Differential gene expression analyses were performed using 669
edgeR (http://bioinf.wehi.edu.au/edgeR/) (Robinson et al., 2010). We selected genes 670
with fold change >2 and P <0.0001 compared to wild type as differential expression 671
genes and TEs. The distribution of differentially-expressed genes and TEs in the 672
chromosomes was plotted by circos (Krzywinski et al., 2009). The categories of 673
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
33
up-regulated TEs were divided as previously described (Wang et al., 2015). 674
Chromatin Immunoprecipitation (ChIP) Assays 675
Nuclei were isolated from 15-day seedlings and fixed with 1% formaldehyde, 676
following the protocol as described previously (Saleh et al., 2008). The pure nuclei 677
were resuspended with 300 µL of cold nuclei lysis buffer, then the genomic DNA was 678
sonicated into 250-500 bp fragments, and the supernatant was diluted with ChIP 679
dilution buffer. Twenty microliters of protein A/G Magnetic beads (Millipore: 16-663) 680
was added for 90 minutes at 4°C to decrease nonspecific combination with gentle 681
rotation. All steps that needed to collect beads were carried out on a Magnetic rack on 682
ice. The antibody H3K9me2 (Abcam: ab1220) was added with gentle rotation over 683
night at 4°C to allow combination. The Magnetic beads were washed five times: one 684
time with low salt wash buffer, one time with high salt wash buffer, one time with 685
LiCl wash buffer and two times with TE buffer. Each wash was 5 minutes at 4°C with 686
gentle rotation. The protein and DNA complex were eluted by elution buffer at 65°C 687
and then incubated at 65°C for at least 6 h or overnight to reverse cross-linking. Next, 688
the RNAs were digested using RNase A at 37°C for 2 hours, and then the proteinase K 689
was added to digest the protein at 65°C at least for 6 hours. The QIAquick PCR 690
Purification Kit (QIAGEN: 28106) was used to obtain high quality DNA, then the 691
concentrations of the DNA measured with Qubit Fluorometer 3.0 (Invitrogen: Q33216) 692
were adjusted to 50 pg/µL. Lastly, 1 µL DNA was used as the template in 20 µL of 693
SYBR Green Master mix (TaKaRa) on a Step One Plus machine (Applied 694
Biosystems). The experiments were performed in three independent biological 695
replicates. 696
Measurement of SAM Contents by liquid chromatography-mass spectrometry 697
(LC-MS) 698
Sixteen-day-old seedlings were used for the subsequent measurements. The extraction 699
and determination methods were followed as previously described with some 700
modifications to extraction (Nikiforova et al., 2005). We added 300 µL methanol 701
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
34
(volume/volume: 80% and precooled at -20°C) and 100 µL precooled methanol with 702
15 mg/mL DTT to 100 mg plant samples that had been ground into powder in liquid 703
nitrogen, vortexed for 1 minute, extracted for 30 minutes on ice and then centrifuged 704
at 4 °C for 10 minutes at 12000 g. We added 300 µL precooled isopropanol and 100 705
µL precooled methanol with 15 mg/mL DTT to re-suspend the precipitation, extracted 706
for 30 minutes on ice and then centrifuged at 4°C for 10 minutes at 12000 g. The two 707
supernatants were combined, filtered, and then analyzed via LC-MS. The experiments 708
were performed in three independent biological replicates with technical triplicate. 709
Acquisition of Knockout Mutants using the CRISPR/Cas9 Assay 710
The targets were selected according to the website of 711
http://www.crisprscan.org/?page=sequence. Two targets were chosen for each gene, 712
and primers were designed. Fragments were amplified by PCR using pCBC-DT1T2 713
as a template (Wang et al., 2015). After PCR products were purified, they were 714
digested using the restriction enzyme BsaI and ligated using T4 ligase in one system 715
for 5 hours at 37°C, 5 minutes at 50°C and 10 minutes at 80°C. Then transformed 716
them into the competent cell of JM109. After incubation, the correct clone was 717
identified. The construct in A. tumefaciens strain GV3101 was transformed into L119 718
by the floral dip method (Clough and Bent, 1998). The transgenic lines were selected 719
on MS medium supplemented with 30 mg/L hygromycin from the T1 generation and 720
sequenced to obtain knockout lines. 721
Determination of the Activities of MATs 722
The activities of MATs were determined by measuring the production amount of SAM 723
after reactions. The purified proteins of MAT1-His, MAT2-His, MAT3-His, 724
MAT4-His and MAT4 (D246N)-His in Escherichia coli were desalted using a 10-kD 725
Centrifugal Filter Unit (Millipore UFC501096). The reaction was carried out in a 200 726
µl mixture that included 40 µg MAT, 10 mM ATP, 5 mM methionine, 0.1 M Tris-HCl 727
(pH=8.0), 0.02 M MgCl2 and 0.2 M KCl at 37°C for 20 minutes, and then the reaction 728
was terminated by adding 800 µl of 75% acetonitrile and 1.2% formic acid. The 729
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
35
reaction solution was transferred to a 10-kD Centrifugal Filter Unit and centrifuged at 730
full speed in 4°C for 10 minutes. The solution was used for LC-MS analysis. The 731
experiments were performed in three independent biological replicates with technical 732
triplicate. 733
Pull-Down Assays 734
The full-length cDNA of MAT1, MAT2, MAT3 and MAT4 fused with His and GST 735
tags were constructed in PET30a and pGEX-4T-1. The relevant plasmids were 736
co-transformed into the Rosetta (DE3) strain of E. coli. The Glutathione-Sepharose 737
beads were used to purify the proteins. Then the proteins were eluted from the 738
Glutathione-Sepharose beads using 10 mM reduced GSH in 50 mM Tris∙HCl. The cell 739
lysates before addition of Glutathione-Sepharose beads were used as input to detect 740
whether two proteins were both expressed. Then products were detected by 741
immunoblot using the antibodies of His and GST. 742
Co-immunoprecipitation (co-IP) Assays in Arabidopsis protoplasts 743
The full-length cDNAs of MAT1, MAT2, MAT3 and MAT4 fused with FLAG and GFP 744
tags under the control of the super promoter were constructed in the pCAMBIA 1300 745
vector. The plasmids were extracted and purified with the Plasmid Maxprep Kit 746
(VIGOROUS N001). Then, the two relevant plasmids were co-introduced into 747
Arabidopsis protoplasts as previously described (Kong et al., 2015). After 14-16 h of 748
incubation in light, the protoplasts were collected and total protein was extracted 749
using the immunoprecipitation buffer (50 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 150 750
mM NaCl, 10% glycerol, 0.5% NP-40, 1 mM DTT, 1 mM PMSF, and protease 751
inhibitor cocktail, 1:100, 1 plate per mL, Roche) for 30 minutes on ice. The protein 752
solution was centrifuged at 12000 g for 15 minutes at 4 °C. Ten microliter 753
GFP-Trap_A (Chromotek: gta-20) was added to the supernatant, then gently rotated 754
for 2.5 hours at 4°C to allow combination. The GFP-Trap_A was washed five times 755
using the immunoprecipitation buffer, then the immunoprecipitated products were 756
detected by immunoblot using the antibodies of GFP and FLAG. 757
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
36
Gel Filtration Assay 758
MAT4-FLAG was used to prepare the protein for gel filtration. More than 20 g of 759
MAT4-FLAG seedlings grown on MS medium for 15 days were collected and then 760
ground to a powder in liquid nitrogen. The protein was extracted using IP buffer (10 761
mM HEPES, 1 mM EDTA (pH=8.0), 100 mM NaCl, 10% glycerol, 0.5%Triton X-100, 762
1 mM DTT, 1 mM PMSF, and protease inhibitor cocktail,1 plate per mL, Roche) for 763
30 minutes on ice. Then the protein solution was centrifuged at 12000 g for 15 764
minutes at 4 °C. The supernatant was added to the ANTI-FLAG M1 Agarose Affinity 765
Gel (Sigma-Aldrich: A4596), then gently rotated for 2.5 hours at 4°C to allow 766
combination. The FLAG Agarose was washed five times using the IP buffer, and the 767
protein was eluted using 0.5 µg/µL FLAG Peptide (Sigma-Aldrich: F3290). We 768
prepared 0.5 mg protein for gel filtration analysis. The effluent of the indicated peaks 769
were sent for LC-MS analysis. 770
Accession numbers 771
The gene accession numbers that were used in this study are as follows: AT3G17390 772
(MAT4/SAM3/MTO3), AT1G02500 (MAT1/SAM1), AT4G01850 (MAT2/SAM2), 773
AT2G36880 (MAT3), DDM1 (AT5G66750), AT3G18780 (ACTIN), AT5G52310 774
(RD29A), AT2G17690 (SDC), AT4G03650 (AtGP1), BD298459.1 (TSIs). 775
RNA-seq, and BS-seq data were deposited in the National Center for Biotechnology 776
Information GEO database under accession number GSE84014. 777
Supplemental Data 778
The following supplemental materials are available. 779
Supplemental Figure S1. T-DNA insertion positions in L119. 780
Supplemental Figure S2. Growth phenotypes of mat4 mutants and Kan-resistant 781
phenotypes of two ddm1 alleles. 782
Supplemental Figure S3. Effects of mat4 on DNA methylation throughout the five 783
chromosomes. 784
Supplemental Figure S4. Complementation of reduced histone modification in 785
mat4 by MAT4-FLAG. 786
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
37
Supplemental Figure S5. Confirmation of RNA-seq data by RT-qPCR and 787
association between DNA methylation and gene expression. 788
Supplemental Figure S6. T-DNA insertion positions, expression levels, and SAM 789
contents in mat1, mat2 and mat3. 790
Supplemental Figure S7. Whole genomic DNA methylation changes in mat1, 791
mat2 and mat3. 792
Supplemental Figure S8. Kanamycin sensitivity of mat1, mat2 and mat3 793
CRISPR/Cas9 mutants in L119. 794
Supplemental Figure S9. Complementation of mat4 by MAT4 driven by the 795
native MAT1 promoter. 796
Supplemental Figure S10. Growth and development phenotypes of mat1mat2 797
double mutants and embryogenic defects of mat2-c19mat4 double mutants. 798
Supplemental Figure S11. The interaction of MAT1, MAT2 and MAT3 with 799
different MATs as determined by co-immunoprecipitation assays. 800
Supplemental Figure S12. The interaction of MAT1, MAT2, MAT3 with different 801
MATs as determined by protein pull-down assays. 802
Supplemental Table 1. LC-MS/MS analyses of affinity co-purified proteins from 803
MAT4-FLAG seedlings. 804
Supplemental Table 2. Primers used in this study 805
Supplemental data set S1. Hypo- differentially methylated regions of CG in mat4. 806
Supplemental data set S2. Hypo- differentially methylated regions of CHG in 807
mat4. 808
Supplemental data set S3. Hypo- differentially methylated regions of CHH in 809
mat4. 810
Supplemental data set S4. Differentially-expressed genes up-regulated in mat4. 811
Supplemental data set S5. Differentially-expressed genes down-regulated in 812
mat4. 813
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
38
Supplemental data set S6. Differentially-expressed TEs up-regulated in mat4. 814
Supplemental data set S7. Differentially-expressed TEs down-regulated in mat4. 815
ACKNOWLEDGEMENTS 816
We thank Dr. Zhen Li and Dr. Zhongzhou Chen in China Agricultural University for 817
assistance in LC-MS analyses and gel filtration, respectively. This study was 818
supported by the Natural Science Foundation of China (31330041). 819
820
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
39
Figure Legends 821
Figure 1. Diagram of the methyl-group supply in one-carbon metabolism. 822
Enzymes involved in one-carbon metabolism: MAT/SAMS (methionine 823
adenosyltransferase/S-adenosyl-methionine synthetase); MT (methyltransferase); 824
SAHH1/HOG1 (S-adenosyl-homocysteine hydrolase/ homology-dependent gene 825
silencing1); MS (methionine synthase); FPGS (folylpolyglutamate synthetase). FPGS 826
catalyzes the synthesis of 5-CH3-THF-Glun, which provides active methyl group for 827
Hcy for Met synthesis by MS. MAT/SAMS uses Met and ATP as substrates to 828
synthesis SAM, which converts to SAH after methylation reaction of MT. 829
SAHH1/HOG1 can hydrolyze SAH to Hcy. 830
Figure 2. Identification and characterization of MAT4 831
A. Kan resistance of mat4 mutants. Seeds were germinated on Murashige and Skoog 832
(MS) medium or MS supplemented with 25 mg/L Kan. L119 was the transgenic line 833
harboring silenced Pro35S::NPTII and ProRD29A::LUC (proRD29A, an abiotic 834
stress-inducible promoter). ddm1-18 (indicated as ddm1) was selected in the same 835
genetic screening and reactivated both transgenic sites. 836
B. Protein levels of NPTII in L119, mat4 and ddm1 detected by immunoblot. ACTIN 837
was the loading control. 838
C. Transcript levels of transgenic and endogenous loci by real-time RT-qPCR analysis. 839
Transcript levels were normalized to ACTIN2 and relative to L119. Three independent 840
experiments were conducted with similar results. Data are from one experiment with 841
three technical replicates. Error bars are the means ± SD, asterisks indicate significant 842
differences determined by Student’s t-test (* indicates P< 0.05; ** indicates P< 0.01; 843
*** indicates P< 0.001). 844
D. Silenced ProRD29A::LUC reactivation in mat4 and ddm1. Seedlings of L119, mat4 845
and ddm1 were treated with 300 mM NaCl for three hours before detecting the 846
inflorescence signal with a CCD camera (Roper, 1300D). 847
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
40
E. Identification of MAT4 by map-based cloning. There was a G to A mutation, which 848
changed 246-Asp to 246-Asn in AT3G17390. 849
F. Complementation of Kan resistance and delayed-germination in mat4 by MAT4. 850
G. NPTII level restored to the basal level of L119 in MAT4-FLAG as determined by 851
immunoblot analyses using anti-NPTII antibodies. ACTIN was the loading control. 852
H. Subcellular localization of MAT4. a. Transgenic line carrying Pro35S::MAT4-GFP 853
in L119; b. Transient expression of ProMAT4::MAT4-GFP in a protoplast; c. 854
Transient expression of ProMAT4::MAT4-GFP in Nicotiana benthamiana leaf 855
epidermal cells. 856
I. Detection of the subcellular localization of MAT4-FLAG after isolating the cytosol 857
and nuclei. PEPC was a marker protein in the cytosol and H3 was a marker protein in 858
the nuclei. 859
Figure 3. DNA methylation of the transgenic and endogenous RD29A promoter 860
in mat4 861
A. DNA methylation of the 35S promoter region by bisulfite sequencing in L119, 862
mat4, ddm1, and MAT4-FLAG. 863
B. DNA methylation of the transgenic RD29A promoter region by bisulfite 864
sequencing in L119, mat4, ddm1, and MAT4-FLAG. 865
C. DNA methylation of the endogenous RD29A promoter region by bisulfite 866
sequencing in L119, mat4, ddm1, and MAT4-FLAG. 867
D. DNA methylation of the T-DNA insertion region in L119 and mat4 as determined 868
by whole-genome bisulfite sequencing as indicated by IGV software windows. 869
Figure 4. Whole-genome DNA methylation levels in mat4 870
A. Whole-genome DNA methylation levels of CG, CHG, and CHH in L119, ddm1 871
and mat4. Bisulfite sequencing data for L119 and mat4 were from this study. Data for 872
ddm1 were from a previously published study (Zemach et al., 2013). 873
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
41
B. Relative changes in the DNA methylation levels of CG, CHG, and CHH in L119, 874
ddm1 and mat4. 875
C. Frequency distribution histograms of significant methylation differences (P< 0.01) 876
between L119 and mat4 in CG, CHG, and CHH. The histograms were made with 877
100-bp analyzable windows over the genome-wide scale and the methylation levels of 878
L119 and mat4 in CG, CHG and CHH context were calculated separately. 879
D. CG, CHG and CHH methylation of L119, ddm1 and mat4 at genes that do not 880
contain TEs (including 2 kb upstream and downstream). TSS: transcription start site; 881
TTS: transcription termination site. 882
E. CG, CHG and CHH methylation of L119, ddm1 and mat4 at TEs that are shorter 883
than 0.5 kb (S-TE), including 2 kb upstream and downstream, and at TE body regions. 884
F. CG, CHG and CHH methylation of L119, ddm1 and mat4 at TEs that are longer 885
than 4 kb (L-TE), including 2 kb upstream and downstream, and at TE body regions. 886
Figure 5. Histone H3K9me2 and H3K27me1 levels in mat4 887
A. Immunoblot assays with antibodies against H3K9me1, H3K9me2, and H3K27me1 888
in L119, ddm1 and mat4. H3 was the loading control. 889
B. Statistical analyses of relative signal intensity in (A). We set the signal intensity of 890
L119 as 100 to calculate the relative signal intensity of other mutants. Error bars are 891
the means ± SD (n=3). Asterisks indicate significant differences determined by 892
Student’s t-test (* indicates P< 0.05; ** indicates P< 0.01). 893
C. Histone methylation patterns of H3K9me1 in the nuclei of L119, ddm1 and mat4 as 894
detected by immunofluorescence assay. 895
D. Histone methylation patterns of H3K9me2 in the nuclei of L119, ddm1 and mat4 as 896
detected by immunofluorescence assay. 897
E. Histone methylation patterns of H3K27me1 in the nuclei of L119, ddm1 and mat4 898
as detected by immunofluorescence assay. 899
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
42
From C-E. On the right, the graphs show the percentage of nuclei with condensed or 900
dispersed signal: gray represents a condensed and white represents a dispersed signal. 901
n = number of nuclei. 4',6-diamidino-2-phenylindole (DAPI) stains the 902
pericentromeric heterochromatin regions. 903
F. Detection of H3K9me2 in L119, ddm1 and mat4 at several selected loci by 904
chromatin immunoprecipitation (ChIP) combined with RT-qPCR. Three independent 905
experiments were conducted with similar results. Data are from one experiment with 906
three technical replicates. Error bars are the means ± SD (n=3). Asterisks indicate 907
significant differences determined by Student’s t-test (* indicates P < 0.05; ** 908
indicates P < 0.01). 909
Figure 6. Gene expression changes in mat4 by RNA sequencing 910
A. Differentially expressed genes in mat4 compared with L119. Transcript levels of 911
genes that changed more than two-fold and had P < 0.0001 were selected. Gene up: 912
up-regulated genes; Gene down: down-regulated genes. 913
B. Differentially expressed TEs in mat4 compared with L119. Transcript levels of TEs 914
that changed more than two-fold and had a P< 0.0001 were selected. TE up: 915
up-regulated TEs; TE down: down-regulated TEs 916
C. Distribution of the differentially expressed genes and TEs on the five chromosomes. 917
The purple circle represents the differentially expressed genes, the blue circle 918
represents the differentially expressed TEs, and the green circle represents the 919
differentially methylated regions in mat4. The outer bars indicate the up-regulated 920
genes, TEs and hyper-differentially methylated regions (DMRs), and the inner bars 921
indicate the down-regulated genes, TEs and hypo-DMRs; the length of the bars 922
represents the fold change of the genes, TEs and DMRs. The black dots indicate the 923
chromocenters. 924
D. Overlap of up-regulated TEs among mat4, ddm1 and fpgs1. The overlap number 925
was calculated using VENNY2.1. 926
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
43
E. Categories of up-regulated TEs in mat4. The diagram shows the percentage of 927
different TE types among the total up-regulated TEs. 928
Figure 7. Application of SAM to rescue the release of silencing in mat4 929
A. SAM content in mat4 compared with L119 as determined by LC-MS. Three 930
independent experiments were conducted with similar results. Data are from one 931
experiment with three technical replicates. Error bars are the means ± SD, n=3. 932
Asterisks indicate significant differences determined by Student’s t-test (** indicates 933
P< 0.01). 934
B. SAH content in mat4 compared with L119 as determined by LC-MS. Three 935
independent experiments were conducted with similar results. Data are from one 936
experiment with three technical replicates. Error bars are the means ± SD, n=3. 937
Asterisks indicate significant differences determined by Student’s t-test (*** indicates 938
P< 0.001). 939
C. Kanamycin resistance of mat4 can be partially rescued by exogenously adding 400 940
mg/L SAM to medium supplemented with 25 mg/L Kan. 941
D. Statistical results show the survival rate of seedlings grown on the indicated 942
medium. Error bars are the means ± SD (n=15). Asterisks indicate significant 943
differences determined by Student’s t-test (* indicates P< 0.05; *** indicates P< 944
0.001). 945
E. Transcript levels of NTPII and endogenous loci by real-time RT-qPCR analysis 946
using the seedlings grown on medium supplemented with 400 mg/L SAM. Three 947
independent experiments were conducted with similar results. Data are from one 948
experiment with three technical replicates. Error bars are the means ± SD. Asterisks 949
indicate significant differences determined by Student’s t-test (* indicates P< 0.05; ** 950
indicates P< 0.01; *** indicates P < 0.001). 951
F. Histone methylation patterns of H3K9me2 in L119 and mat4 seedlings grown on 952
MS medium or MS medium supplemented with 400 mg/L SAM as determined by 953
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
44
immunofluorescence assays with anti-H3K9me2 antibodies. DAPI staining (blue) was 954
performed on the pericentromeric heterochromatin regions. 955
G. The percentage of nuclei that showed a condensed or dispersed signal. n = number 956
of nuclei. 957
Figure 8. The catalytic activities of MAT proteins 958
A. Comparison of the catalytic activities of MAT proteins. The same amount of MAT 959
proteins as indicated by Coomassie staining were added for individual reactions. The 960
reaction that had no protein added was used as a negative control. Three independent 961
experiments were conducted with similar results. Data are from one experiment with 962
three technical replicates. Error bars are the means ± SD (n=3). 963
B. SAM production with increasing concentrations of MAT4. Protein amounts were 964
indicated by Coomassie staining. The reaction that had no protein added was used as a 965
negative control. Three independent experiments were conducted with similar results. 966
Data are from one experiment with three technical replicates. Error bars are the means 967
± SD (n=3). 968
C, D. The GFP fluorescence of mat4 transgenic lines carrying the MAT4 promoter 969
driving MAT1, MAT2 or MAT3 cDNA. The seedlings were grown on MS for seven 970
days, and the GFP fluorescence in root tips (C) or the whole seedlings (D) was 971
visualized by a confocal microscope (Zeiss LSM 510 META) and a fluorescent 972
microscope (Olympus SEX16), respectively. 973
E. Kan resistance and delayed-germination in mat4 was complemented by MAT1, 974
MAT2 or MAT3 driven by the promoter of MAT4. 975
Figure 9. MAT4 plays a predominant role in plant growth and development 976
A. Kan resistance and delayed-germination in mat4 was not complemented by 977
ProMAT1::MAT4-GFP, ProMAT2:: MAT4-GFP or ProMAT3::MAT4-GFP, but was 978
complemented by ProMAT4::MAT2-GFP. 979
B. The GFP fluorescence in seedlings of transgenic lines carrying 980
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
45
ProMAT1::MAT4-GFP, ProMAT2::MAT4-GFP, ProMAT3::MAT4-GFP or 981
ProMAT4::MAT2-GFP grown on MS for seven days. 982
C. Statistical results of the fluorescence intensity of the transgenic lines in (B) in a 983
fixed area in cotyledons by ImageJ. Error bars are the mean ± SD (n=12). 984
D. Detection of MAT4-GFP in transgenic lines by immunoblotting using anti-GFP 985
antibodies. ACTIN was the loading control. 986
E. mat1-c19 mat4 double mutant seedlings compared to the wild type (L119) grown in 987
soil under long-day conditions. The mutant did not produce any seeds. 988
F. The siliques of WT and mat2-c13(-/-) mat4(+/-). Asterisks indicate the wizened 989
seeds of mat2-c13 mat4 homozygous double mutants. 990
G. Wizened seed percentages in siliques of mat2-c13(-/-) mat4(+/-) heterozygous 991
mutants compared to the WT. 992
Figure 10. MAT4 interacts with different MATs in plants 993
A. MAT4 interactions with MAT1, MAT2, MAT3 or MAT4 itself in a protein 994
co-immunoprecipitation (co-IP) assay. Total proteins were extracted from Arabidopsis 995
protoplasts transiently co-expressing the MAT4-FLAG with MAT1-, MAT2-, MAT3-, 996
MAT4-GFP or GFP (as a negative control) plasmids, and immunoprecipitated with 997
anti-GFP beads. The co-IP proteins were immunoblotted with anti-FLAG and 998
anti-GFP antibodies. 999
B. Protein pull-down assay for MAT4 interaction with MAT1, MAT2, MAT3 or 1000
MAT4 itself. Total proteins were isolated from E. coli co-expressing MAT4-His with 1001
GST-MAT1, -MAT2, -MAT3, -MAT4 or GST itself (as a negative control), and 1002
immunoprecipitated with Glutathione-Sepharose beads. The co-IP proteins were 1003
immunoblotted with anti-His and anti-GST antibodies. 1004
C. Gel filtration analyses. The 0.5 mg of total proteins extracted from approximately 1005
20 g of the 15-day seedlings of MAT4-FLAG were applied to an ANTI-FLAG M1 1006
Agarose Affinity Gel. The proteins were eluted using 0.5 µg/µL FLAG Peptide. The 1007
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
46
elution at the peaks was used for LC/MS analysis. 1008
D. LC-MS/MS analyses of the proteins of the three peaks in (C). Cov indicated the 1009
percentage of sequence coverage (%), Seq (sig) indicated number of significant 1010
sequences. 1011
1012
1013
1014
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
Parsed CitationsAndrea Steimer PA, Karin Afsar,Paul Fransz,Ortrun Mittelsten Scheid,and Jerzy Paszkowskia (2000) Endogenous targets oftranscriptional gene silencing in Arabidopsis.pdf. The Plant Cell 12: 1165–1178
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Baubec T, Dinh HQ, Pecinka A, Rakic B, Rozhon W, Wohlrab B, von Haeseler A, Mittelsten Scheid O (2010) Cooperation of multiplechromatin modifications can generate unanticipated stability of epigenetic States in Arabidopsis. Plant Cell 22: 34-47
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bernatavichute YV, Zhang X, Cokus S, Pellegrini M, Jacobsen SE (2008) Genome-wide association of histone H3 lysine ninemethylation with CHG DNA methylation in Arabidopsis thaliana. PLoS One 3: e3156
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bruce P. May ZBL, Yuda Fang, David L. Spector, Robert A. Martienssen (2005) Differential Regulation of Strand-Specific Transcriptsfrom Arabidopsis Centromeric Satellite Repeats. PLoS Genetics 1
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cao X, Jacobsen SE (2002) Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferasegenes. Proceedings of the National Academy of Sciences 99: 16491-16498
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cao X, Jacobsen SE (2002) Role of the arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing. Curr Biol12: 1138-1144
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chen Y, Zou T, McCormick S (2016) S-Adenosylmethionine Synthetase 3 Is Important for Pollen Tube Growth. Plant Physiol 172: 244-253
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J16: 735-743
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
De La Haba G, Cantoni GL (1959) The enzymatic synthesis of S-adenosyl-L-homocysteine from adenosine and homocysteine. J BiolChem 234: 603-608
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Du J, Johnson LM, Groth M, Feng S, Hale CJ, Li S, Vashisht AA, Gallego-Bartolome J, Wohlschlegel JA, Patel DJ, Jacobsen SE (2014)Mechanism of DNA methylation-directed histone methylation by KRYPTONITE. Mol Cell 55: 495-504
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Du J, Zhong X, Bernatavichute YV, Stroud H, Feng S, Caro E, Vashisht AA, Terragni J, Chin HG, Tu A, Hetzel J, Wohlschlegel JA,Pradhan S, Patel DJ, Jacobsen SE (2012) Dual binding of chromomethylase domains to H3K9me2-containing nucleosomes directs DNAmethylation in plants. Cell 151: 167-180
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ebbs ML, Bartee L, Bender J (2005) H3 lysine 9 methylation is maintained on a transcribed inverted repeat by combined action ofSUVH6 and SUVH4 methyltransferases. Mol Cell Biol 25: 10507-10515
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
Ebbs ML, Bender J (2006) Locus-specific control of DNA methylation by the Arabidopsis SUVH5 histone methyltransferase. Plant Cell18: 1166-1176
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Friso S, Choi SW, Girelli D, Mason JB, Dolnikowski GG, Bagley PJ, Olivieri O, Jacques PF, Rosenberg IH, Corrocher R, Selhub J (2002)A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interactionwith folate status. Proc Natl Acad Sci U S A 99: 5606-5611
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fusao Takusagawa SK, and George D. Markham (1996) Structure and Function of S-Adenosylmethionine Synthetase Crystal Structuresof S-Adenosylmethionine Synthetase with ADP, BrADP, and PPi at 2.8 Å Resolution.pdf. Biochemistry 35: 2586-2596
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Goto DB, Ogi M, Kijima F, Kumagai T, van Werven F, Onouchi H, Naito S (2002) A single-nucleotide mutation in a gene encoding S-adenosylmethionine synthetase is associated with methionine over-accumulation phenotype in Arabidopsis thaliana. Genes & GeneticSystems 77: 89-95
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Groth M, Moissiard G, Wirtz M, Wang H, Garcia-Salinas C, Ramos-Parra PA, Bischof S, Feng S, Cokus SJ, John A, Smith DC, Zhai J,Hale CJ, Long JA, Hell R, Diaz de la Garza RI, Jacobsen SE (2016) MTHFD1 controls DNA methylation in Arabidopsis. Nat Commun 7:11640
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
He XJ, Hsu YF, Pontes O, Zhu J, Lu J, Bressan RA, Pikaard C, Wang CS, Zhu JK (2009) NRPD4, a protein related to the RPB4 subunit ofRNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA-directed DNA methylation. Genes Dev 23: 318-330
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
He XJ, Hsu YF, Pontes O, Zhu JH, Lu J, Bressan RA, Pikaard C, Wang CS, Zhu JK (2009) NRPD4, a protein related to the RPB4 subunitof RNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA-directed DNA methylation. Genes &Development 23: 318-330
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
He XJ, Hsu YF, Zhu S, Wierzbicki AT, Pontes O, Pikaard CS, Liu HL, Wang CS, Jin H, Zhu JK (2009) An effector of RNA-directed DNAmethylation in arabidopsis is an ARGONAUTE 4- and RNA-binding protein. Cell 137: 498-508
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Henderson IR, Jacobsen SE (2008) Tandem repeats upstream of the Arabidopsis endogene SDC recruit non-CG DNA methylation andinitiate siRNA spreading. Genes Dev 22: 1597-1606
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jackson JP, Lindroth AM, Cao X, Jacobsen SE (2002) Control of CpNpG DNA methylation by the KRYPTONITE histone H3methyltransferase. Nature 416: 556-560
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jacob Y, Feng S, LeBlanc CA, Bernatavichute YV, Stroud H, Cokus S, Johnson LM, Pellegrini M, Jacobsen SE, Michaels SD (2009)ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing. Nat Struct Mol Biol 16:763-768
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jin Y, Ye N, Zhu F, Li H, Wang J, Jiang L, Zhang J (2017) Calcium-dependent protein kinase CPK28 targets the methionineadenosyltransferases for degradation by the 26S proteasome and affects ethylene biosynthesis and lignin deposition in Arabidopsis.Plant J www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from
Copyright © 2018 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Johnson LM, Bostick M, Zhang X, Kraft E, Henderson I, Callis J, Jacobsen SE (2007) The SRA methyl-cytosine-binding domain linksDNA and histone methylation. Curr Biol 17: 379-384
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kanno T, Bucher E, Daxinger L, Huettel B, Bohmdorfer G, Gregor W, Kreil DP, Matzke M, Matzke AJM (2008) A structural-maintenance-of-chromosomes hinge domain-containing protein is required for RNA-directed DNA methylation. Nature Genetics 40: 670-675
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kong L, Cheng J, Zhu Y, Ding Y, Meng J, Chen Z, Xie Q, Guo Y, Li J, Yang S, Gong Z (2015) Degradation of the ABA co-receptor ABI1 byPUB12/13 U-box E3 ligases. Nat Commun 6: 8630
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Krueger F, Andrews SR (2011) Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27: 1571-1572
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA (2009) Circos: an information aesthetic forcomparative genomics. Genome Res 19: 1639-1645
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the humangenome. Genome Biol 10: R25
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11:204-220
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Li W, Han Y, Tao F, Chong K (2011) Knockdown of SAMS genes encoding S-adenosyl-l-methionine synthetases causes methylationalterations of DNAs and histones and leads to late flowering in rice. J Plant Physiol 168: 1837-1843
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Li X, Qian W, Zhao Y, Wang C, Shen J, Zhu JK, Gong Z (2012) Antisilencing role of the RNA-directed DNA methylation pathway and ahistone acetyltransferase in Arabidopsis. Proc Natl Acad Sci U S A 109: 11425-11430
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lindermayr C, Saalbach G, Bahnweg G, Durner J (2006) Differential inhibition of Arabidopsis methionine adenosyltransferases byprotein S-nitrosylation. J Biol Chem 281: 4285-4291
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Mao D, Yu F, Li J, Van de Poel B, Tan D, Li J, Liu Y, Li X, Dong M, Chen L, Li D, Luan S (2015) FERONIA receptor kinase interacts withS-adenosylmethionine synthetase and suppresses S-adenosylmethionine production and ethylene biosynthesis in Arabidopsis. PlantCell Environ 38: 2566-2574
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Mao DD, Yu F, Li J, Van de Poel B, Tan D, Li JL, Liu YQ, Li XS, Dong MQ, Chen LB, Li DP, Luan S (2015) FERONIA receptor kinaseinteracts with S-adenosylmethionine synthetase and suppresses S-adenosylmethionine production and ethylene biosynthesis inArabidopsis. Plant Cell and Environment 38: 2566-2574
Pubmed: Author and Title www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from
Copyright © 2018 American Society of Plant Biologists. All rights reserved.
CrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Matzke MA, Kanno T, Matzke AJ (2015) RNA-Directed DNA Methylation: The Evolution of a Complex Epigenetic Pathway in FloweringPlants. Annu Rev Plant Biol 66: 243-267
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Mehrshahi P, Gonzalez-Jorge S, Akhtar TA, Ward JL, Santoyo-Castelazo A, Marcus SE, Lara-Nunez A, Ravanel S, Hawkins ND, BealeMH, Barrett DA, Knox JP, Gregory JF, 3rd, Hanson AD, Bennett MJ, Dellapenna D (2010) Functional analysis of folate polyglutamylationand its essential role in plant metabolism and development. Plant J 64: 267-279
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Mentch SJ, Mehrmohamadi M, Huang L, Liu X, Gupta D, Mattocks D, Gomez Padilla P, Ables G, Bamman MM, Thalacker-Mercer AE,Nichenametla SN, Locasale JW (2015) Histone Methylation Dynamics and Gene Regulation Occur through the Sensing of One-CarbonMetabolism. Cell Metab 22: 861-873
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Molloy AM (2012) Genetic aspects of folate metabolism. Subcell Biochem 56: 105-130Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Mull L, Ebbs ML, Bender J (2006) A histone methylation-dependent DNA methylation pathway is uniquely impaired by deficiency inArabidopsis S-adenosylhomocysteine hydrolase. Genetics 174: 1161-1171
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Murray B, Antonyuk SV, Marina A, Van Liempd SM, Lu SC, Mato JM, Hasnain SS, Rojas AL (2014) Structure and function study of thecomplex that synthesizes S-adenosylmethionine. IUCrJ 1: 240-249
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nikiforova VJ, Kopka J, Tolstikov V, Fiehn O, Hopkins L, Hawkesford MJ, Hesse H, Hoefgen R (2005) Systems rebalancing ofmetabolism in response to sulfur deprivation, as revealed by metabolome analysis of Arabidopsis plants. Plant Physiol 138: 304-318
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ouyang B, Fei Z, Joung JG, Kolenovsky A, Koh C, Nowak J, Caplan A, Keller WA, Cui Y, Cutler AJ, Tsang EW (2012) Transcriptomeprofiling and methyl homeostasis of an Arabidopsis mutant deficient in S-adenosylhomocysteine hydrolase1 (SAHH1). Plant Mol Biol 79:315-331
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Peleman J, Boerjan W, Engler G, Seurinck J, Botterman J, Alliotte T, Van Montagu M, Inze D (1989) Strong cellular preference in theexpression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase. Plant Cell 1: 81-93
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Peleman J, Saito K, Cottyn B, Engler G, Seurinck J, Van Montagu M, Inze D (1989) Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana. Gene 84: 359-369
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ravanel S, Block MA, Rippert P, Jabrin S, Curien G, Rebeille F, Douce R (2004) Methionine metabolism in plants: chloroplasts areautonomous for de novo methionine synthesis and can import S-adenosylmethionine from the cytosol. J Biol Chem 279: 22548-22557
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital geneexpression data. Bioinformatics 26: 139-140
Pubmed: Author and TitleCrossRef: Author and Title
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
Google Scholar: Author Only Title Only Author and Title
Rocha PS, Sheikh M, Melchiorre R, Fagard M, Boutet S, Loach R, Moffatt B, Wagner C, Vaucheret H, Furner I (2005) The ArabidopsisHOMOLOGY-DEPENDENT GENE SILENCING1 gene codes for an S-adenosyl-L-homocysteine hydrolase required for DNAmethylation-dependent gene silencing. Plant Cell 17: 404-417
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Saleh A, Alvarez-Venegas R, Avramova Z (2008) An efficient chromatin immunoprecipitation (ChIP) protocol for studying histonemodifications in Arabidopsis plants. Nat Protoc 3: 1018-1025
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sauter M, Moffatt B, Saechao MC, Hell R, Wirtz M (2013) Methionine salvage and S-adenosylmethionine: essential links betweensulfur, ethylene and polyamine biosynthesis. Biochem J 451: 145-154
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Shen B, Li C, Tarczynski MC (2002) High free-methionine and decreased lignin content result from a mutation in the Arabidopsis S-adenosyl-L-methionine synthetase 3 gene. Plant J 29: 371-380
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Shyh-Chang N, Locasale JW, Lyssiotis CA, Zheng Y, Teo RY, Ratanasirintrawoot S, Zhang J, Onder T, Unternaehrer JJ, Zhu H, AsaraJM, Daley GQ, Cantley LC (2013) Influence of threonine metabolism on S-adenosylmethionine and histone methylation. Science 339:222-226
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Singer T, Yordan C, Martienssen RA (2001) Robertson's Mutator transposons in A. thaliana are regulated by the chromatin-remodelinggene Decrease in DNA Methylation (DDM1). Genes Dev 15: 591-602
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Soppe WJ, Jasencakova Z, Houben A, Kakutani T, Meister A, Huang MS, Jacobsen SE, Schubert I, Fransz PF (2002) DNA methylationcontrols histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis. EMBO J 21: 6549-6559
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Stroud H, Do T, Du JM, Zhong XH, Feng SH, Johnson L, Patel DJ, Jacobsen SE (2014) Non-CG methylation patterns shape theepigenetic landscape in Arabidopsis. Nature Structural & Molecular Biology 21: 64-+
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Stroud H, Greenberg MV, Feng S, Bernatavichute YV, Jacobsen SE (2013) Comprehensive analysis of silencing mutants revealscomplex regulation of the Arabidopsis methylome. Cell 152: 352-364
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tariq M, Saze H, Probst AV, Lichota J, Habu Y, Paszkowski J (2003) Erasure of CpG methylation in Arabidopsis alters patterns ofhistone H3 methylation in heterochromatin. Proc Natl Acad Sci U S A 100: 8823-8827
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Teixeira FK, Heredia F, Sarazin A, Roudier F, Boccara M, Ciaudo C, Cruaud C, Poulain J, Berdasco M, Fraga MF, Voinnet O, Wincker P,Esteller M, Colot V (2009) A role for RNAi in the selective correction of DNA methylation defects. Science 323: 1600-1604
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Thomas D, Rothstein R, Rosenberg N, Surdin-Kerjan Y (1988) SAM2 encodes the second methionine S-adenosyl transferase inSaccharomyces cerevisiae: physiology and regulation of both enzymes. Mol Cell Biol 8: 5132-5139
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
Thomas D, Surdin-Kerjan Y (1987) SAM1, the structural gene for one of the S-adenosylmethionine synthetases in Saccharomycescerevisiae. Sequence and expression. J Biol Chem 262: 16704-16709
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Thomas D, Surdin-Kerjan Y (1991) The synthesis of the two S-adenosyl-methionine synthetases is differently regulated inSaccharomyces cerevisiae. Mol Gen Genet 226: 224-232
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang C, Dong X, Jin D, Zhao Y, Xie S, Li X, He X, Lang Z, Lai J, Zhu JK, Gong Z (2015) Methyl-CpG-binding domain protein MBD7 isrequired for active DNA demethylation in Arabidopsis. Plant Physiol 167: 905-914
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang ZP, Xing HL, Dong L, Zhang HY, Han CY, Wang XC, Chen QJ (2015) Egg cell-specific promoter-controlled CRISPR/Cas9efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biology 16
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xia R, Wang J, Liu C, Wang Y, Wang Y, Zhai J, Liu J, Hong X, Cao X, Zhu JK, Gong Z (2006) ROR1/RPA2A, a putative replication proteinA2, functions in epigenetic gene silencing and in regulation of meristem development in Arabidopsis. Plant Cell 18: 85-103
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yamaguchishinozaki K, Shinozaki K (1994) A Novel Cis-Acting Element in an Arabidopsis Gene Is Involved in Responsiveness toDrought, Low-Temperature, or High-Salt Stress. Plant Cell 6: 251-264
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yang DL, Zhang G, Tang K, Li J, Yang L, Huang H, Zhang H, Zhu JK (2016) Dicer-independent RNA-directed DNA methylation inArabidopsis. Cell Res 26: 66-82
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ye R, Chen Z, Lian B, Rowley MJ, Xia N, Chai J, Li Y, He XJ, Wierzbicki AT, Qi Y (2016) A Dicer-Independent Route for Biogenesis ofsiRNAs that Direct DNA Methylation in Arabidopsis. Mol Cell 61: 222-235
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zemach A, Kim MY, Hsieh P-H, Coleman-Derr D, Eshed-Williams L, Thao K, Harmer Stacey L, Zilberman D (2013) The ArabidopsisNucleosome Remodeler DDM1 Allows DNA Methyltransferases to Access H1-Containing Heterochromatin. Cell 153: 193-205
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang H, Deng X, Miki D, Cutler S, La H, Hou YJ, Oh J, Zhu JK (2012) Sulfamethazine suppresses epigenetic silencing in Arabidopsisby impairing folate synthesis. Plant Cell 24: 1230-1241
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhao Y, Xie S, Li X, Wang C, Chen Z, Lai J, Gong Z (2014) REPRESSOR OF SILENCING5 Encodes a Member of the Small Heat ShockProtein Family and Is Required for DNA Demethylation in Arabidopsis. Plant Cell 26: 2660-2675
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhou HR, Zhang FF, Ma ZY, Huang HW, Jiang L, Cai T, Zhu JK, Zhang C, He XJ (2013) Folate polyglutamylation is involved in chromatinsilencing by maintaining global DNA methylation and histone H3K9 dimethylation in Arabidopsis. Plant Cell 25: 2545-2559
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
www.plantphysiol.orgon April 9, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.