METHIONINE ADENOSYLTRANSFERASE4 Mediates · MAT4 knockout mutations generated by CRISPR/Cas9 were lethal, indicating that MAT4 is an essential gene in Arabidopsis. MAT1, 2, and 4

METHIONINE ADENOSYLTRANSFERASE4 MediatesDNA and Histone Methylation1[OPEN]

Jingjing Meng,2 Lishuan Wang,2 Jingyi Wang, Xiaowen Zhao, Jinkui Cheng, Wenxiang Yu, Dan Jin,Qing Li, and Zhizhong Gong3

State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China AgriculturalUniversity, Beijing 100193, China

ORCID IDs: 0000-0001-6994-1704 (L.W.); 0000-0001-6551-6014 (Z.G.).

DNA and histone methylation coregulate heterochromatin formation and gene silencing in animals and plants. To identifyfactors involved in maintaining gene silencing, we conducted a forward genetic screen for mutants that release the silencedtransgene Pro35S::NEOMYCIN PHOSPHOTRANSFERASE II in the transgenic Arabidopsis (Arabidopsis thaliana) line L119. Weidentified MAT4/SAMS3/MTO3/AT3G17390, which encodes methionine (Met) adenosyltransferase 4 (MAT4)/S-adenosyl-Metsynthetase 3 that catalyzes the synthesis of S-adenosyl-Met (SAM) in the one-carbon metabolism cycle. mat4 mostly decreasesCHG and CHH DNA methylation and histone H3K9me2 and reactivates certain silenced transposons. The exogenous additionof SAM partially rescues the epigenetic defects of mat4. SAM content and DNA methylation were reduced more in mat4 than inthree othermatmutants.MAT4 knockout mutations generated by CRISPR/Cas9 were lethal, indicating thatMAT4 is an essentialgene in Arabidopsis. MAT1, 2, and 4 proteins exhibited nearly equal activity in an in vitro assay, whereas MAT3 exhibitedhigher activity. The native MAT4 promoter driving MAT1, 2, and 3 cDNA complemented the mat4 mutant. However, most mat4transgenic lines carrying nativeMAT1, 2, and 3 promoters drivingMAT4 cDNA did not complement the mat4mutant because oftheir lower expression in seedlings. Genetic analyses indicated that the mat1mat4 double mutant is dwarfed and the mat2mat4double mutant was nonviable, while mat1mat2 showed normal growth and fertility. These results indicate that MAT4 plays apredominant role in SAM production, plant growth, and development. Our findings provide direct evidence of the cooperativeactions between metabolism and epigenetic regulation.

DNA and histone methylation are important epige-netic modifications that regulate gene expression andgenome stability, and can be inherited (Law and Jacob-sen, 2010). Compared with animals, which largely dis-play CG methylation, plants present symmetric CG andCHGmethylation and asymmetric CHHmethylation. InArabidopsis (Arabidopsis thaliana), DNA methylation ismediated by the RNA-directed DNA methylation path-way (RdDM) and dicer-independent RdDM (Matzkeet al., 2015; Yang et al., 2016; Ye et al., 2016); CG meth-ylation is maintained by DNA METHYLTRANSFER-ASE1 (MET1; a functional equivalent protein of DNAmethyltransferase 1 in mammals); CHH methylation is

maintained by DOMAINS REARRANGED METHYL-ASE2 (DRM2) and CHROMOMETHYLASE2 (CMT2;Cao and Jacobsen, 2002a, 2002b;Du et al., 2014); andCHGmethylation is maintained by CMT3. CHGmethylation isrecognized by the SET and RING Associated (SRA) do-main histone methyltransferase KRYPTONITE/SU(VAR)homolog 4 (SUVH4) and its homologs SUVH5 andSUVH6 to establish the dimethylation of histone H3 atLys 9 (H3K9me2) (Jackson et al., 2002; Ebbs et al., 2005;Ebbs and Bender, 2006). H3K9me2 is bound by CMT3through its H3 tails (Johnson et al., 2007; Bernatavichuteet al., 2008; Law and Jacobsen, 2010; Du et al., 2012),which form a reinforcing feedback loop that maintainsCHG methylation and H3K9me2.

The one-carbon metabolism pathway plays an im-portant role in epigenetic regulation because it providesmethyl groups for most methylation reactions (Fig. 1).The initial methyl group donor is poly-Glu-5-methyl-tetrahydrofolate (5-CH3-THF-Glun), which is the mostcommon form of folate and has a high affinity for folate-dependent methionine (Met) synthase as the methyl-group donor. Folate-dependent Met synthase catalyzesthe methylation of homocysteine (Hcy) to Met using5-CH3-THF-Glun as the methyl-group donor (Friso et al.,2002; Ravanel et al., 2004; Mehrshahi et al., 2010).S-adenosyl-Met (SAM), one of the most abundant co-factors in plant metabolism, is synthesized by Metadenosyltransferase (MAT; also known as S-adenosyl-Met synthetase [SAMS]) using Met and ATP as

1 This study was supported by the National Natural ScienceFoundation of China (31330041).

2 These authors contributed equally to the article.3 Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Zhizhong Gong ([email protected]).

Z.G. conceived the original research plans; J.M. performed most ofthe experiments; LW. performed the mutant screening and gene clon-ing; J.C. provided bioinformatics analysis; X.Z., J.W., D.J., W.Y., andQ.L. assisted with some experiments. J.M. and Z.G. designed theproject and wrote the article with contributions from all the authors.

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substrates. After transferring a methyl group to DNA,RNA, proteins or other metabolites by SAM-dependentmethyltransferases (Sauter et al., 2013), SAM is changedinto S-adenosyl-Hcy (SAH), which competes with SAMand is an inhibitor for many Met synthetases (Molloy,2012). SAH is then converted to adenosine and Hcy bySAH hydrolase encoded by the HOMOLOGY-DEPENDENT GENE SILENCING1 (HOG1) in Ara-bidopsis, thus finishing a single cycle of one-carbonmetabolism. The T-DNA (hog1-5) or transposon (hog1-4)insertion mutants are zygotic embryo lethal, whereasits weak mutation can cause delayed germination,poor growth, reduced seed viability, and reducedwhole-genome DNA and histone methylation (Rochaet al., 2005; Mull et al., 2006; Baubec et al., 2010; Ouyanget al., 2012). A mutation of folylpolyglutamate synthe-tase 1 (FPGS1) that converts 5-CH3-THF-Glu1 to 5-CH3-THF-Glun in Arabidopsis can slow germination andreduce levels of whole-genome DNA methylationand H3K9me2 (Zhou et al., 2013). Treatment with

sulfamethazine, which is a structural analog and com-petitor of p-aminobenzoic acid, the precursor of folate,causes the release of endogenous transposons andrepeat elements and the reduction of DNA methyl-ation levels and H3K9me2 (Zhang et al., 2012). Amutation in the cytoplasmic bifunctional methylenete-trahydrofolate dehydrogenase/methenyltetrahydrofolatecyclohydrolase (MTHFD1) leads to decreased levels ofoxidized tetrahydrofolates, DNA hypomethylation, loss ofH3K9me, and transposon reactivation (Groth et al., 2016).

The Arabidopsis genome has four MAT genes withdifferent nomenclatures (MAT1/SAM1/AT1G02500,MAT2/SAM2/AT4G01850, MAT3/AT2G36880, andMAT4/MTO3/SAMS3/AT3G17390) in different pub-lications (Peleman et al., 1989a, 1989b; Goto et al., 2002;Mao et al., 2015; Chen et al., 2016). A previous studyindicates that SAMS RNAi transgenic rice (Oryza sativa)lines with down-regulation ofOsSAMS1, 2, and 3 showreduced histone H3K4me3 and DNA methylation (Liet al., 2011). In Arabidopsis, the pollen expressedMAT3

Figure 1. Diagram of the methyl-group supply in one-carbon metabolism. Enzymes involved in one-carbon metabolism: MAT/SAMS, Met adenosyltransferase/S-adenosyl-Met synthetase; MT, methyltransferase; SAHH1/HOG1, S-adenosyl-homo-cysteinehydrolase/ homology-dependent gene silencing1; MS, Met synthase; and FPGS, folylpolyglutamate synthetase. FPGS catalyzesthe synthesis of 5-CH3-THF-Glun, which provides active methyl group for Hcy for Met synthesis by MS. MAT/SAMS uses Met andATPas substrates to synthesis SAM,which converts to SAH aftermethylation reaction ofMT. SAHH1/HOG1 can hydrolyze SAH toHcy.

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MAT4 Mediates DNA and Histone Methylation

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is required for maintaining histone and tRNA methyla-tion in pollen, pollen germination, andpollen tube growth(Chen et al., 2016). However, the biological roles of otherMAT proteins in Arabidopsis epigenetic regulation arestill unknown.

In this study, we screened a mutagenized populationfrom the transgenic line L119, which harbors two si-lenced transgenes, Pro35S::NPTII (NEOMYCIN PHOS-PHOTRANSFERASE II) and ProRD29A (RESPONSETO DESSICATION 29A; a stress-inducible promoter)::LUC, and identified the mat4/sams3/mto3 (Met over-accumulation) mutant (Shen et al., 2002; Jin et al., 2017)that releases the silencing of both genes. We found thatthe mat4 mutant, harboring a missense point mutation,dramatically decreases SAM content and CHG andCHH methylation and H3K9me2, leading to the acti-vation of some transposable elements. Exogenous ad-ditions of SAM to themediumpartially restored histonemethylation levels in mat4. The mat1, 2, or 3 mutantsreduced SAM content and DNAmethylation to a lesserextent than did mat4, indicating a predominant role ofMAT4 among MATs. MAT3 showed the highest ac-tivity among the fourMAT proteins in an in vitro assay.The expression of MAT4 in seedlings was much higherthan MAT1, MAT2, and MAT3. The MAT4 promoterdriving MAT1, MAT2, or MAT3 cDNA could comple-ment the mat4 mutant, while most transgenic linescarrying theMAT1,MAT2, orMAT3 promoters drivingMAT4 cDNA could not complement the mat4 mutant.The MAT4 loss-of-function mutation generated usingthe CRISPR/Cas9 technique was lethal. We also foundthat the MAT proteins in Arabidopsis interacted witheach other and themselves both in vitro and in vivo,indicating that they may form homologous or hetero-geneous oligomers in Arabidopsis.

RESULTS

Identification and Characterization of mat4

To further study the mechanisms regulating tran-scriptional gene silencing, we obtained the transgenicline L119 carrying ProRD29A::LUC and Pro35S::NPTIIin the Columbia gl1 background. In L119, the transgeneloci consist of at least two T-DNA insertions, each withtwo repeats (Supplemental Fig. S1, A–F). ProRD29A is astress-inducible promoter that is induced by abscisicacid, low temperatures, and high NaCl concentrations(Yamaguchi-Shinozaki and Shinozaki, 1994). The L119plants were very sensitive to kanamycin (Kan) andshowed little luciferase activity; they grew poorly onmedia containing 25 mg/L Kan (Fig. 2A) and did notemit any fluorescence after NaCl treatment (Fig. 2D),indicating that both ProRD29A::LUC and Pro35S::NPTIIare silenced in L119. However, after introducing thedefective in meristem silencing 3 (dms3-1) mutation in theRdDM pathway (Kanno et al., 2008) into L119,ProRD29A::LUC, but not Pro35S::NPTII, was reactivated(Supplemental Fig. S1, G–J), suggesting that similar to theC24/RD29A::LUC line (He et al., 2009a), ProRD29A::LUC

is regulated by the RdDM pathway, while Pro35S::NPTIIis not.

The transgenic line L119 was mutagenized by EMS,and the F2 population was screened for Kan-resistantmutants. A mutant, named mat4-3, was isolated in thisscreen (hereafter referred to as mat4; Fig. 2A). mat4seeds germinated later (Fig. 2A) and the seedlings weresmaller compared with L119, although these seedlingshad relatively normal fertility (Supplemental Fig. S2, Aand B). Two alleles of ddm1, ddm1-18 and ddm1-19, werealso identified in this system. ddm1-18 (a G-to-A changeat position 2803 [counting from the first putative ATGin the coding frame], which causes a stop codon with aTGG-to-TGA transition; hereafter referred to as ddm1)and ddm1-19 (a G-to-A change at position 3125, whichcauses a stop codon with a TGG-to-TGA transition)exhibited more resistance to Kan than mat4, whereasL119 seedlings did not survive on the medium con-taining 25 mg/L Kan (Supplemental Fig. S2C). DDM1 isa nucleosome-remodeling protein involved in facilitat-ingDNAmethyltransferase access to heterochromatin tosilence certain transposable elements and repeats in co-operation with the RdDM pathway (Singer et al., 2001;Zemach et al., 2013). Here, we used ddm1 as a positivecontrol for reduced DNAmethylation. Greater increasesofNPTII expression and its protein levels were observedinmat4 than in L119, although theNPTII expression andprotein levels were less in mat4 than in ddm1 (Fig. 2, Band C). Treatment with 300 mM NaCl reactivated theexpression of ProRD29A::LUC in mat4 (Fig. 2D). Twosilenced transgenic loci were reactivated in mat4, whichwas also observed in ddm1; thus, we detected endoge-nously silenced genes, DNA repeats, and transposableelements. SUPPRESSOROF DRM1DRM2 CMT3 (SDC)is regulated by non-CG DNA methylation (Hendersonand Jacobsen, 2008); TSIs are endogenous transcrip-tionally silent information sites regulated by the DNAreplication and repair pathway and DNA methylationindependent of the RdDM pathway (Steimer et al., 2000;Xia et al., 2006); AtGP1 is a long terminal repeat-Gypsytransposonmodulated by the RdDM pathway (He et al.,2009a, 2009b); 180-bp CEN is a centromeric satellite re-peat regulated by DNA methylation independent of theRdDM pathway but not by the DNA replication andrepair pathway (May et al., 2005; Xia et al., 2006). Thetranscript levels of all these loci were higher inmat4 thanin L119 but lower than in ddm1 (Fig. 2C).

We then cloned the MAT4 gene by map-based clon-ing. We first crossed the mat4 mutant with the wild-type Ler. The 519 F2 plants that were Kan resistantwere isolated and used for mapping. We narrowedmat4 to a region between bacterial artificial chromo-some clones K14A17 and MPK6 on chromosome 3. Wesequenced candidate genes in this region and observedthat a G-to-A mutation in AT3G17390 changed 246D to246N (Fig. 2E; Supplemental Fig. S2D). This mutationoccurs in a conserved amino acid that is involved inbinding Met during the reaction, according to thecrystal structure of human (Homo sapiens) MAT2A,which is different from the mto3 mutation in the ATP

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binding site (Shen et al., 2002). This point mutation didnot cause alteration in the transcript level (SupplementalFig. S2E). To confirm whether the mutation in MAT4 isresponsible for the Kan resistance of mat4, we trans-formed the full genomic length of MAT4, including the2,221-bp promoter and the full genomic sequence fusedwith FLAG or a GFP tag into themat4mutant. A numberof different transgenic lines were obtained and shown tobe Kan sensitive, which was also observed in L119. Weselected one MAT4-FLAG line (line 1) for further study

(hereafter referred as MAT4-FLAG). Immunoblottingusing anti-FLAG antibodies indicated that MAT4-FLAGexpressed the MAT4-FLAG protein (Supplemental Fig.S2F).MAT4-FLAGwas sensitive to Kan and had normalgermination (Fig. 2F), and the NPTII protein level wasrestored to the basal level observed in L119 (Fig. 2G),suggesting that AT3G17390 could complement the mat4mutant phenotype. Reverse transcription quantitativePCR (RT-qPCR) analyses indicated that the expression ofNPTII and certain endogenous loci in the MAT4-FLAG

Figure 2. Identification and characterizationofMAT4.A,Kan resistance ofmat4mutants. Seedswere germinatedonMSmediumorMSsupplemented with 25 mg/L Kan. L119was the transgenic line harboring silenced Pro35S::NPTII and ProRD29A::LUC (proRD29A, anabiotic stress-inducible promoter). ddm1-18 (indicated as ddm1) was selected in the same genetic screening and reactivated bothtransgenic sites. B, Protein levels of NPTII in L119, mat4, and ddm1 detected by immunoblot. ACTIN was the loading control.C, Transcript levels of transgenic and endogenous loci by real-time RT-qPCR analysis. Transcript levels were normalized toACTIN2 andrelative to L119. Three independent experimentswere conductedwith similar results.Data are fromone experimentwith three technicalreplicates. Error bars are the means6 SD; asterisks indicate significant differences determined by Student’s t test (*P, 0.05, **P, 0.01,***P , 0.001). D, Silenced ProRD29A::LUC reactivation in mat4 and ddm1. Seedlings of L119, mat4, and ddm1 were treated with300 mM NaCl for 3 h before detecting the inflorescence signal with a CCD camera (Roper 1300D). E, Identification ofMAT4 by map-based cloning. Therewas aG-to-Amutation,which changedAsp-246 toAsn-246 inAT3G17390. F, Complementation of Kan resistanceand delayed germination in mat4 by MAT4. G, NPTII level restored to the basal level of L119 in MAT4-FLAG as determined by im-munoblot analyses using anti-NPTII antibodies. ACTINwas the loading control. H, Subcellular localization ofMAT4. a, Transgenic linecarrying Pro35S::MAT4-GFP in L119; b, transient expression of ProMAT4::MAT4-GFP in a protoplast; c, transient expression ofProMAT4::MAT4-GFP inN. benthamiana leaf epidermal cells. I, Detection of the subcellular localization of MAT4-FLAG after isolatingthe cytosol and nuclei. PEPC was a marker protein in the cytosol and H3 was a marker protein in the nuclei.









also returned to the basal level observed in L119(Supplemental Fig. S2G). We did not observe any en-hanced severe phenotypes of the mat4 mutant afterseveral generations. We used the egg cell-specificCRISPR/Cas9 system in L119 (Wang et al., 2015b) andcreated two MAT4 knockout lines, mat4-c19 and mat4-c32. mat4-c19 has a fragment deletion from 76 to 180 bp(counting from the first putative ATG) and mat4-c32from 706 to 797 bp (Supplemental Fig. S2H). However,we were unable to obtain homozygous mat4 mutants,indicating thatMAT4 is an essential gene inArabidopsis.

The subcellular localization in transgenic L119 plantsexpressing Pro35S::MAT4-GFP indicated that MAT4-GFP was localized in the nucleus and cytosol (Fig. 2H,a). A similar localization ofMAT4-GFPwas observed ina transient transformation assay using Arabidopsisprotoplasts andNicotiana benthamiana leaves (Fig. 2H, band c). To avoid the possibility that GFP translocates tothe nucleus by itself, we isolated the cytosol and nucleifrom L119 and the MAT4-FLAG transgenic line andperformed an immunoblot assay. MAT4-FLAG proteinwas detected in both the cytosol and the nucleus (Fig.2I). Previous studies also indicate that SAM1/MAT1,SAM2/MAT2, and MAT3 are all localized in both cy-tosol and nuclei (Mao et al., 2015; Chen et al., 2016).These results suggest that MAT proteins may functionin both the cytosol and the nucleus in Arabidopsis.

DNA Methylation of Transgenic Loci Is Reduced in mat4

MAT4 catalyzes the biosynthesis of SAM, which is auniversal methyl group donor for DNA and histone

methylation; thus, mat4 may reactivate the silencedPro-35S::NPTII and ProRD29A::LUC because of decreasedDNA and/or histone methylation. Bisulfite sequencinganalyses indicated that CHG and CHH methylation oftransgenic and endogenous RD29A promoters largelydecreased in mat4 compared with that in L119 (Fig. 3, Band C), which was consistent with the data from whole-genome bisulfite sequencing (Fig. 3D). However, CHGand CHH methylation decreased to a lesser extent at the35S promoter in mat4 compared with that in L119 (Fig. 3,A and D). However, only limited changes were observedin the CG methylation with transgenic RD29A and 35Spromoters in mat4. In contrast, ddm1 greatly reduced CGmethylation and only moderately affected CHG andCHH methylation, except for the transgene RD29A pro-moter, in which ddm1 did not affect CHHmethylation. Inaddition, the DNA methylation of the MAT4-FLAG linewas consistently restored to the L119 level (Fig. 3, A–C).These results indicate that mat4 reduces DNA methyla-tion in transgenes in L119.

mat4 Reduces DNA Methylation at the Whole-Genome Level

We compared the DNA methylation level of mat4with that of L119 at the whole-genome level by bisulfitesequencing. We obtained nearly 5G raw data includingadapter and low-quality data for each sample, fromwhich we obtained 4.2G clean data for our subsequentanalyses. The total readsweremapped to the genome ofTAIR10. We then obtained the methylation level of CG,CHG, and CHH by calculating the ratio of C to C+C/T

Figure 3. DNA methylation of thetransgenic and endogenous RD29Apromoter in mat4. A, DNA methyla-tion of the 35S promoter regionby bisulfite sequencing in L119,mat4, ddm1, and MAT4-FLAG. B,DNA methylation of the transgenicRD29A promoter region by bisulfitesequencing in L119,mat4, ddm1, andMAT4-FLAG. C, DNA methylation ofthe endogenous RD29A promoter re-gion by bisulfite sequencing in L119,mat4, ddm1, and MAT4-FLAG. D,DNA methylation of the T-DNA inser-tion region in L119 and mat4 as de-termined by whole-genome bisulfitesequencing as indicated by IGV soft-ware windows.


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using the tool of Bismark (Krueger and Andrews, 2011;Fig. 4A). We also included the previously publisheddata of ddm1 (Zemach et al., 2013) for comparison. Themethylation levels of CG (22.9%), CHG (4.2%), andCHH (1.4%) in mat4 were lower than the levels of CG(25.2%), CHG (8.2%), and CHH (2.4%) in L119 (Fig. 4A).CHG and CHH methylation in mat4 decreased bynearly half, whereas CG methylation decreased ap-proximately by 9.2%, suggesting thatmat4 has different

effects on CG, CHG, and CHH DNA methylation (Fig.4B).mat4 displayed a relatively smaller reduction of CGand CHG methylation and greater reduction of CHHmethylation compared with that observed in ddm1(Fig. 4B). Frequency distribution histograms of sig-nificant methylation differences between L119 andmat4 in CG, CHG, and CHH also indicated that theCHG and CHHmethylation dramatically decreased inmat4 (Fig. 4C).

Figure 4. Whole-genomeDNAmethylation levels inmat4. A,Whole-genomeDNAmethylation levels of CG, CHG, and CHH inL119, ddm1, andmat4. Bisulfite sequencing data for L119 andmat4were from this study. Data for ddm1were from a previouslypublished study (Zemach et al., 2013). B, Relative changes in the DNAmethylation levels of CG, CHG, and CHH in L119, ddm1,andmat4. C, Frequency distribution histograms of significant methylation differences (P, 0.01) between L119 andmat4 in CG,CHG, and CHH. The histograms were made with 100-bp analyzable windows over the genome-wide scale and the methylationlevels of L119 andmat4 in CG, CHG, and CHH context were calculated separately. D, CG, CHG, and CHHmethylation of L119,ddm1, and mat4 at genes that do not contain TEs (including 2 kb upstream and downstream). TSS, Transcription start site; TTS,transcription termination site. E, CG, CHG, and CHH methylation of L119, ddm1, and mat4 at TEs that are shorter than 0.5 kb(S-TE), including 2 kb upstream and downstream, and at TE body regions. F, CG, CHG, and CHHmethylation of L119, ddm1, andmat4 at TEs that are longer than 4 kb (L-TE), including 2 kb upstream and downstream, and at TE body regions.





To determine the distribution of changes in the DNAmethylation patterns in detail, we calculated the DNAmethylation 2 kb upstream and downstream of thegenes and transposable elements (TEs), respectively. Ingenes that exclude TEs or repeats, CG methylationmainly occurred in the gene bodies. mat4 reduced theCG methylation in the gene body regions by ;2.5%(Fig. 4D). However, ddm1 showed greater reductions inCG methylation compared with mat4 in these regions(Fig. 4D). CHG and CHH methylation did not shownoticeable changes (Fig. 4D) because these regions ex-hibit limited CHG and CHH methylation. For TEs, wefocused on two TE types: TEs shorter than 0.5 kb (S-TEs;usually regulated by the RdDM pathway) and TEs lon-ger than 4 kb (L-TEs; usually regulated by the DDM1pathway; Teixeira et al., 2009; Zemach et al., 2013).Generally, mat4 displayed less CG, CHG, and CHHmethylation than the L119 in both S-TEs and L-TEs (Fig.4, E and F). Compared with ddm1, mat4 displayed moreCG methylation in both short and long TEs and moreCHG methylation in long TEs. However, mat4 showedsimilar CHG methylation in short TEs and CHH meth-ylation in long TEs as ddm1 (Fig. 4, E and F). mat4 dis-played less CHH methylation in short TEs than ddm1,which is consistent with previous studies of DDM1regulation of DNA methylation in long TEs, but notshort TEs that are mostly regulated by the RdDM path-way (Teixeira et al., 2009; Zemach et al., 2013). These re-sults suggest that mat4 mainly reduces CHG and CHHmethylation and, to a lesser extent, CG methylation.When mapping these hypo-differentially methylated re-gions (hypo-DMRs) to the five chromosomes, we foundthat the distribution of these hypo-DMRs were concen-trated around the five centromeres, which displayed adramatic decrease of CHG and CHH methylation(Supplemental Fig. S3; Supplemental Data Sets S1–S3).These results indicate that mat4 reduces genomic-wideDNA methylation, especially CHG and CHH methyla-tion at pericentromeric heterochromatin regions.

mat4 Decreases Histone Modifications inHeterochromatin Regions

Because DNA methylation, especially CHG and CHHmethylation, is reduced throughout the genome in mat4,we sought to determine whether mat4 has an effect onhistone methylation. We verified the histone modifica-tions in H3K9me2, H3K9me1, and H3K27me1 becausethese modifications usually accompany DNA methyla-tion in heterochromatin regions, and we also verified themodifications in H3K4me3 because this modification ac-companies high gene expression (Tariq et al., 2003; Jacobet al., 2009). Using immunoblotting assays, we found thatthe H3K9me2 levels in mat4were comparable to those inddm1 and greatly reduced comparedwith that of L119. Inaddition, only a small decrease in H3K9me1 was exhibi-ted inmat4, whereas a dramatic decreasewas observed inddm1 compared with L119 (Fig. 5, A and B). Both mat4and ddm1 had a lower H3K27me1 level than L119. In the

mat4 complementary line, H3K9me2, H3K9me1, andH3K27me1 were restored to the wild-type level, whereasin mat4, ddm1, L119, or MAT4-FLAG, H3K4me3 was notchanged (Supplemental Fig. S4, A and B).

We then compared the heterochromatin status in nu-clei using an immunofluorescence assay with differentantibodies. In the wild-type cells, more than 89% ofthe interphase nuclei showed H3K9me2, H3K9me1,and H3K27me1 immunofluorescence associated withthe condensed pericentromeric heterochromatin regionsstained with 49,6-diamidino-2-phenylindole (DAPI).However,;78% ofmat4 and 87% of ddm1 nuclei showedchromocenter decondensation and reduced H3K9me2immunofluorescence. ddm1 showed strongly reducedH3K9me1 immunofluorescence, which was not ob-served inmat4, whereas ddm1 andmat4mutants showedsubstantially reduced H3K27me1 immunofluorescence(Fig. 5, C–E). In MAT4-FLAG, H3K9me2 immunofluo-rescence was restored to the L119 level (SupplementalFig. S4, C and D). However, we did not detect a differ-ence in H3K4me3 immunofluorescence in L119, mat4,and MAT4-FLAG (Supplemental Fig. S4, E and F). Weconfirmed the decrease of H3K9me2 at certain loci usingchromatin immunoprecipitation (ChIP)-PCR (Fig. 5F).These results suggest that mat4 reduces the histonemethylation in heterochromatin regions, especiallyH3K9me2 and H3K27me1.

mat4 Reactivates Silenced TEs

To determine how mat4 modulates gene expression,we performed RNA sequencing (RNA-seq). Total RNAwas extracted from 15-d-old seedlings and then sub-jected to RNA-seq with two biological replicates. Weobtained 3G clean data with each replicate, mapped allof the obtained reads to TAIR10, and then compared thetranscript levels between mat4 and L119 using edgeR(Robinson et al., 2010). We obtained 1284 protein codinggenes and 364 TEs with transcript level changes of atleast 2-fold and P, 0.0001. Among these protein-codinggenes, 66% (842) were up-regulated and 34% (442) weredown-regulated (Fig. 6,A andB; SupplementalData SetsS4–S7). After mapping these genes on the five chromo-somes, we found that theywere evenly distributed alongthe chromosomes arms and rarely localized at the cen-tromere regions (Fig. 6C). Approximately 92% (334) ofthe differentially expressed TEs were up-regulated andconcentrated around the centromeres (Fig. 6C). The ex-pression of certain genes and TEs was confirmed byRT-qPCR in L119,mat4, andMAT4-FLAG (SupplementalFig. S5, A and B). Compared with previously publisheddata in ddm1 and fpgs1, we found that 127 up-regulatedTEs in mat4 were also up-regulated in ddm1 and fpgs1with reduced DNAmethylation and H3K9me2 (Fig. 6D;Zemach et al., 2013; Zhou et al., 2013). After dividing theup-regulated TEs according to their characteristics, twocategories of TEs, long terminal repeat/Gypsy and En-hancer/Suppressor Mutator-like transposons (Fig. 6E),accounted for nearly 50% of all of the up-regulated TEs


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in mat4. Alterations to DNA methylation were not as-sociated with the expression of protein coding genes;however, reduced DNAmethylation inmat4was closelyassociated with increased TE expression (SupplementalFig. S5C). When comparing the CHG hypo-DMRs andup-regulated TEs in mat4 with the published data insuvh4/5/6 and cmt3 (Stroud et al., 2013), we found thatthese mutants had higher overlap than that by chance asviewed in VENNY diagram (Supplemental Fig. S5D),indicating that MAT4 affects a large number of targets

shared with those methyltransferases. In conclusion,mat4 led to the activation of the silenced transposons as aresult of the reduction in DNA methylation and histonemethylation.

Application of SAM Partially Rescues the Phenotype of mat4

To test whether the decreases of DNA and histonemethylation were caused by the alteration of SAMcontent in mat4, we measured SAM contents in mat4,

Figure 5. Histone H3K9me2 and H3K27me1 levels in mat4. A, Immunoblot assays with antibodies against H3K9me1, H3K9me2,and H3K27me1 in L119, ddm1, andmat4. H3 was the loading control. B, Statistical analyses of relative signal intensity in A.We set thesignal intensity of L119 as 100 to calculate the relative signal intensity of other mutants. Error bars are the means6 SD (n = 3). Asterisksindicate significant differences determined by Student’s t test (*P, 0.05 and ** P, 0.01). C, Histone methylation patterns of H3K9me1in the nuclei of L119, ddm1, andmat4 as detected by immunofluorescence assay. D, Histone methylation patterns of H3K9me2 in thenuclei of L119, ddm1, andmat4 as detected by immunofluorescence assay. E,Histonemethylation patterns ofH3K27me1 in the nuclei ofL119, ddm1, andmat4 as detected by immunofluorescence assay. For C to E, on the right, the graphs show the percentage of nuclei withcondensedor dispersed signal; gray represents a condensed, andwhite represents a dispersed signal.n=number of nuclei.DAPI stains thepericentromeric heterochromatin regions. F, Detection of H3K9me2 in L119, ddm1, andmat4 at several selected loci by ChIP combinedwith RT-qPCR. Three independent experiments were conducted with similar results. Data are from one experiment with three technicalreplicates. Error bars are the means6 SD (n = 3). Asterisks indicate significant differences determined by Student’s t test (*P, 0.05 and**P, 0.01).








L119, and MAT4-FLAG by liquid chromatography-mass spectrometry (LC-MS). The SAM content in mat4was decreased by nearly 35% compared to that in L119(Fig. 7A). Interestingly, the content of SAH, which is astrong inhibitor of SAM-dependent methyltransferases,was significantly increased in mat4, leading to a largedecrease in the ratio of SAM/SAH, which is an im-portant index influencing the methylation status (Fig.7B). Meanwhile, the contents of SAM and SAH inMAT4-FLAG were restored to L119 level (Fig. 7, A andB). Therefore, we sought to determine whether the ex-ogenous application of SAM could rescue the pheno-type of mat4. After adding 400 mg/L SAM to themedium, the Kan susceptibility of mat4 was partiallyrescued (Fig. 7, C and D). An analysis of the transcriptlevels of transgenic and endogenous genes indicatedthat the application of SAM inhibited the high ex-pression of these genes in mat4 (Fig. 7E). An immu-nofluorescence assay indicated that H3K9me2 levelswere restored by the application of SAM (Fig. 7, F andG). These results suggest that the decreased SAM in

mat4 leads to the release of the silencing of these testedgenes.

MAT4 Plays a Predominant Role in SAM Production andDNA Methylation among Different MAT Homologs

In Arabidopsis, four MAT homologs present near90% identity between each other in their amino acidsequences (Shen et al., 2002; Lindermayr et al., 2006).Because mat4 reduces SAM content and DNA methyl-ation, we sought to determine whether other MATmutants have similar roles. We obtained three T-DNAlines: SALK_059210 carrying a T-DNA insertion in theC terminus of AT1G02500 (mat1), SALK_052006 carry-ing a T-DNA insertion in the N terminus of AT4G01850(mat2), and SALK_019375 carrying a T-DNA insertionafter the putative stop codon of AT2G36880 (mat3),which was used in the previous study (Chen et al.,2016). All three T-DNA insertion lines greatly reducedthe expression of each targeted gene (Supplemental Fig.S6A). We measured the contents of SAM in these

Figure 6. Gene expression changes inmat4 by RNA sequencing. A, Differentially expressed genes inmat4 compared with L119.Transcript levels of genes that changed more than 2-fold and had P, 0.0001 were selected. Gene up, up-regulated genes; Genedown, down-regulated genes. B, Differentially expressed TEs inmat4 compared with L119. Transcript levels of TEs that changedmore than 2-fold and had a P, 0.0001 were selected. TE up, up-regulated TEs; TE down, down-regulated TEs. C, Distribution ofthe differentially expressed genes and TEs on the five chromosomes. The purple circle represents the differentially expressedgenes, the blue circle represents the differentially expressed TEs, and the green circle represents the differentially methylatedregions in mat4. The outer bars indicate the up-regulated genes, TEs, and hyper-DMRs, and the inner bars indicate the down-regulated genes, TEs, and hypo-DMRs; the length of the bars represents the fold change of the genes, TEs and DMRs. The blackdots indicate the chromocenters. D, Overlap of up-regulated TEs among mat4, ddm1, and fpgs1. The overlap number was cal-culated using VENNY2.1. E. Categories of up-regulated TEs in mat4. The diagram shows the percentage of different TE typesamong the total up-regulated TEs.


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mutants and found that SAM content in mat1 and mat2decreased only about 6% compared to that in L119, butno clear change was observed in mat3 (SupplementalFig. S6B), which is consistent with its main expressionin pollen (Chen et al., 2016). We further measured theDNA methylation level in these mutants at the whole-genome level by bisulfite sequencing and found that theDNA methylation in CG, CHG, and CHH slightly de-creased in these mutants (Supplemental Figure S7A),among which mat3 CHG and CHH methylation was

reduced to a lesser extent thanmat1 or 2 in L- and S-TEs(Supplemental Fig. S7, B and C), which was consistentwith the SAM contents in these mutants. These resultsindicate that MAT1, 2, and 3 have less of an effect onDNA methylation than MAT4 in seedlings. However,MAT3 may play a major role in pollens (Chen et al.,2016).

In order to clarify the effect of these MAT proteins onexpression of Pro35S::NPTII in L119, we created loss offunction mutants ofMAT1, 2, and 3 genes using the egg

Figure 7. Application of SAM to rescue the release of silencing in mat4. A, SAM content in mat4 compared with L119 as de-termined by LC-MS. Three independent experiments were conducted with similar results. Data are from one experiment withthree technical replicates. Error bars are the means6 SD, n = 3. Asterisks indicate significant differences determined by Student’s ttest (**P , 0.01). B, SAH content in mat4 compared with L119 as determined by LC-MS. Three independent experiments wereconductedwith similar results. Data are from one experiment with three technical replicates. Error bars are themeans6 SD, n = 3.Asterisks indicate significant differences determined by Student’s t test (***P, 0.001). C, Kan resistance ofmat4 can be partiallyrescued by exogenously adding 400mg/L SAM tomedium supplementedwith 25mg/L Kan. D, Statistical results show the survivalrate of seedlings grown on the indicatedmedium. Error bars are the means6 SD (n = 15). Asterisks indicate significant differencesdetermined by Student’s t test (*P , 0.05 and ***P , 0.001). E, Transcript levels of NTPII and endogenous loci by real-timeRT-qPCR analysis using the seedlings grown onmedium supplementedwith 400mg/L SAM. Three independent experimentswereconducted with similar results. Data are from one experiment with three technical replicates. Error bars are the means 6 SD.Asterisks indicate significant differences determined by Student’s t test (*P , 0.05, **P , 0.01, and ***P , 0.001). F, Histonemethylation patterns of H3K9me2 in L119 andmat4 seedlings grown onMSmediumorMSmedium supplementedwith 400mg/LSAM as determined by immunofluorescence assays with anti-H3K9me2 antibodies. DAPI staining (blue) was performed on thepericentromeric heterochromatin regions. G, The percentage of nuclei that showed a condensed or dispersed signal. n = numberof nuclei.









cell-specific CRISPR/Cas9 system in the L119 back-ground (Supplemental Fig. S8A; Wang et al., 2015b).mat1-c3was a single base insertion mutant, in which anA insertion after 421 bp (counting from the first putativeATG), led to a frame-shift mutation; mat1-c19 hada fragment deletion from 222 to 450 bp; mat2-c6 was asingle base deletion mutant at 488 bp, leading to aframe-shift mutation; mat2-c13 had a fragment deletionfrom 490 to 569 bp; mat3-c8 had a fragment deletionfrom 366 to 420 bp. The expression of each target genewas greatly reduced compared to the wild type(Supplemental Fig. S8A). All these mutants were sen-sitive to Kan (Supplemental Fig. S8B) and the tran-scriptional levels of NPTII did not differ with that inL119 (Supplemental Fig. S8C), indicating that the mu-tations inMAT 1, 2, and 3 do not release the silencing ofPro35S::NPTII, which was consistent with the results ofa smaller reduction in DNA methylation in theirT-DNA lines. The mat3-c8mutant produced one to twoseeds per silique, which is similar with previous results(Chen et al., 2016).

The Expression Pattern of MAT4 Determines ItsPredominant Biological Roles in Arabidopsis

Although the amino acid sequences of the fourMATsshared a high percentage of identity, these proteins didnot compensate for each other in plants. Whether theirexpression patterns or protein activities determinedtheir specificity is not known. To address this question,first, we compared the catalytic activities of those fourproteins in vitro. The MAT proteins were expressed inand purified from Escherichia coli. MAT1, MAT2, andMAT4 exhibited similar activity, while MAT3 hadhigher activity than other three (Fig. 8A). The amount ofSAM produced increased with increasing MAT4 pro-tein concentration as well (Fig. 8B). However, we couldnot detect any activity for the MAT4D246N mutant pro-tein (Fig. 8B), indicating that the mutant protein largelyloses its activity in vitro.

We used the MAT4 promoter driving MAT1, MAT2,or MAT3 cDNA to evaluate whether these cDNAscould complement mat4. Here, we fused MAT cDNAwith GFP to observe its expression. We obtained12 mat4 transgenic lines carrying ProMAT4::MAT1-GFP, 8 carrying ProMAT4::MAT2-GFP, and 19 carryingProMAT4::MAT3-GFP. These transgenic plants hadhigh GFP fluorescence. All these transgenic plants hadhigh expression of transgenes and complemented mat4mutant phenotypes (Fig. 8, C–E). These results indi-cated that MAT proteins have comparable biologicalfunctions in plants.

A previous study indicated that transformingPro-35S::MAT2 into mto3-1 (the mat4 allele) failed tocomplement the mto3 phenotype (Shen et al., 2002).Given that MAT1 and MAT2 are expressed in mostplant tissues (Peleman et al., 1989a;Mao et al., 2015) andMAT3 is mainly expressed in pollens (Chen et al., 2016),we usedMAT1, 2, or 3 promoters drivingMAT4 cDNA

to examine whether they could complement mat4. Weobtained 27 mat4 transgenic lines carrying ProMAT1::MAT4-GFP, 5 carrying ProMAT2::MAT4-GFP, and6 carrying ProMAT3::MAT4-GFP. We found that allProMAT2::MAT4-GFP or ProMAT3::MAT4-GFP andmost ProMAT1::MAT4-GFP transgenic plants did notcomplement the mat4 Kan-resistant phenotype becausethey had lower GFP levels, as indicated by fluorescenceimaging and immunoblotting using GFP antibodies (Fig.9, A–D). In contrast, ProMAT4::MAT2-GFP transgeniclines hadhigherGFP levels (Fig. 9,A–D). In 27ProMAT1::MAT4-GFP transgenic lines, 7 lines showed differentKan-sensitive phenotypes. We selected three lines andcompared their Kan sensitivity with other lines. Thesethree lines showed more Kan sensitivity than other lines(Supplemental Fig. S9A), indicating that they com-plemented or partially complemented the mat4 mutant.GFP protein levels were higher in these complementedlines than in noncomplemented lines as indicated byGFPfluorescence and protein immunoblotting (SupplementalFig. S9, B–D). The expression levels of transgenes werealso higher in these complemented lines than others(Supplemental Fig. S9E). The higher expression of Pro-MAT1::MAT4-GFP may be caused by the different ge-nomic site in which the T-DNA was inserted. Theseresults indicate that the expression level of MAT genesdetermined their biological roles in Arabidopsis.

We performed further genetic analyses among thesemutants. mat1mat2 double mutants did not show anygrowth or developmental differences from the wild-type plants (Supplemental Fig. S10A). mat1-c19 mat4double mutant seedlings were much smaller than thewild type and did not produce any seeds (Fig. 9E). Wecould not obtain mat2-c13 mat4 homozygous doublemutants because the double mutants had embryonicdefects (Fig. 9, F and G; Supplemental Fig. S10B). Theseresults indicate that the expression pattern of MAT4gene determines its predominant biological roles inArabidopsis.

MATs Form Homologous or Heterologous Oligomersin Cells

To explore the functions of MAT4, we tried to iden-tify the MAT4-interacting proteins by immunoprecipi-tation followed by mass spectrometry analysis usingthe complementary transgenic line MAT4-FLAG. Weprecipitated MAT4-FLAG with anti-FLAG beads andused the L119 lines as negative controls. We identifiedMAT1, MAT2, and MAT3, each with unique peptidesfrom MAT4-FLAG coimmunoprecipitation (co-IP)proteins (Supplemental Table S1). Then, we confirmedtheir interactions in vivo using coimmunoprecipitationassays in Arabidopsis protoplasts transiently express-ing different proteins (Fig. 10A). In E. coli, when coex-pressing MAT4-His with GST-MAT1, GST-MAT2,GST-MAT3, GST-MAT4, or only GST (as the negativecontrol), respectively, we found that each of them, butnot GST, could be purified together with MAT4-His


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(Fig. 10B), suggesting that MAT4 can interact withMAT1, MAT2, MAT3, and itself in vitro. We furtherconfirmed that MAT1, MAT2, and MAT3 were able tointeract with each other and themselves in both in vivoand in vitro assays (Supplemental Figs. S11 and S12).Next, we carried out gel filtration using the proteinsisolated from the MAT4-FLAG transgenic comple-mentary line. We observed three peaks from the elutedfractions (Fig. 10C). LC-MS analyses of each peak au-thenticated four MAT proteins (Fig. 10D), indicatingthat theseMATs can formdifferent sizes of homologousor heterologous oligomer complexes in vivo, whichmerits further examination.

DISCUSSION

SAM provides methyl groups for numerous meth-yltransferases in transmethylation reactions, includingDNA and histone methylations in all living cells. In thisstudy, we identified MAT4 because its mutation reac-tivates the silenced Pro35S::NPTII and ProRD29A::LUCin L119. MAT is well conserved during evolution, and it

usually has three domains: the N-terminal domain, theC-terminal domain, and the central M-domain(Takusagawa et al., 1996). In Arabidopsis, there are fourhomologs of MAT, MAT1 to MAT4; these homologsshare nearly 90% amino acid sequence identity(Peleman et al., 1989a, 1989b; Shen et al., 2002).mto3, anallele of mat4, was isolated in a screen based on ethio-nine (a toxic analog of Met) sensitivity. The level of Metis increasedmore than 200-fold and the concentration ofSAM is decreased by 35% comparedwith the wide type(Shen et al., 2002). In this study, we found that bothDNA and histone methylation were largely reduced asa result of the decrease of SAM content in mat4. Ourstudy provides direct evidence for the importance ofSAM in providing methyl donors and modulatingepigenetic status.

In theory, SAM is a general methyl group donor andthe reduction of SAM should have an unbiased effect onDNA and histone methylations. However, we foundthat the reduction of DNA and histonemethylationwasuneven, withmat4 showing large decreases in CHG andCHHmethylation as well as H3K9me2 and H3K27me1

Figure 8. The catalytic activities ofMAT proteins. A, Comparison of thecatalytic activities of MAT proteins.The same amount of MAT proteins asindicated by Coomassie stainingwere added for individual reactions.The reaction that had no proteinadded was used as a negative con-trol. Three independent experimentswere conducted with similar results.Data are from one experiment withthree technical replicates. Error barsare the means 6 SD (n = 3). B, SAMproduction with increasing concen-trations of MAT4. Protein amountswere indicated by Coomassie stain-ing. The reaction that had no proteinadded was used as a negative con-trol. Three independent experimentswere conducted with similar results.Data are from one experiment withthree technical replicates. Error barsare the means6 SD (n = 3). C and D,The GFP fluorescence of mat4transgenic lines carrying the MAT4promoter driving MAT1, MAT2, orMAT3 cDNA. The seedlings weregrown on MS for 7 d, and the GFPfluorescence in root tips (C) or thewhole seedlings (D) was visualizedby a confocal microscope (ZeissLSM 510 META) and a fluorescentmicroscope (Olympus SEX16), re-spectively. E, Kan resistance anddelayed germination in mat4 wascomplemented by MAT1, MAT2, orMAT3 driven by the promoter ofMAT4.






(Figs. 4 and 5). Changes in H3K4me3were not observedand CG methylation decreased to a lesser extent. Inanimals, the supplementation of Met, an essentialamino acid, can modulate the SAM/SAH ratio andimpact H3K4me3 (Mentch et al., 2015). Threonine (Thr),another essential amino acid, is themajor fuel source forglycine, acetyl-CoA, and SAM. Restricted applicationsof Thr can reduce H3K4me3 levels, which results inslower growth and increased differentiation in mouseembryonic stem cells (Shyh-Chang et al., 2013). In ad-dition, in SAMS RNAi rice, H3K4me3 is significantlyreduced (Li et al., 2011). These results suggest that SAMlimitation can result in different changes in DNA andhistone modifications in different species. These dif-ferences can be explained by several factors. First, dif-ferent methyltransferases might have different SAMconcentration thresholds. In addition, MET1 and H3K4

methyltransferases might efficiently use low concen-trations of SAM to complete the reactions in mat4,whereas the histone methyltransferases for H3K9me2and DNAmethyltransferases for CHG and CHHmighthave lower activity at such concentrations. Second, thereinforcing loop between CHG methylation andH3K9me2 cannot be maintained and is even disruptedin mat4, which would lead to a serious reduction inmethylation for both. Third, the increased SAH in mat4would compete with SAM and decrease SAM accessi-bility to methyltransferases, which mostly reduced theCHG and CHH methylation and H3K9me. Similar re-sults have been observed in both fpgs1 and mthfd1mutants (Zhou et al., 2013; Groth et al., 2016). Both fpgs1and mthfd1 mutants accumulate relatively more SAH,which leads to a decreased ratio of SAM/SAH (Zhouet al., 2013; Groth et al., 2016). SAH is a strong inhibitor

Figure 9. MAT4 plays a predominant role in plant growth and development. A, Kan resistance and delayed germination inmat4was not complemented by ProMAT1::MAT4-GFP, ProMAT2:: MAT4-GFP, or ProMAT3::MAT4-GFP but was complemented byProMAT4::MAT2-GFP. B, The GFP fluorescence in seedlings of transgenic lines carrying ProMAT1::MAT4-GFP, ProMAT2::MAT4-GFP, ProMAT3::MAT4-GFP, or ProMAT4::MAT2-GFP grown on MS for 7 d. C, Statistical results of the fluorescence in-tensity of the transgenic lines in B in a fixed area in cotyledons by ImageJ. Error bars are the mean6 SD (n = 12). D, Detection ofMAT4-GFP in transgenic lines by immunoblotting using anti-GFP antibodies. ACTIN was the loading control. E, mat1-c19 mat4double mutant seedlings compared to the wild type (L119) grown in soil under long-day conditions. The mutant did not produceany seeds. F, The siliques of the wild type andmat2-c13(2/2)mat4(+/2). Asterisks indicate the wizened seeds ofmat2-c13 mat4homozygous double mutants. G, Wizened seed percentages in siliques of mat2-c13(2/2) mat4(+/2) heterozygous mutantscompared to the wild type.


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that competes with SAM for SAM-dependent trans-methylation (De La Haba and Cantoni, 1959). Thesestudies suggest that the CMT3 or the CMT2 pathwayhas a positive feedback circuit with SUVH4 (KRYP-TONITE)/5/6 to maintain CHG or CHHmethylationand H3K9me (Zhou et al., 2013; Stroud et al., 2014;Groth et al., 2016). Consistent with the reduced DNAmethylation, our RNA-seq data indicated that a largenumber of TEs were activated in the mat4 mutant.These up-regulated TEs were enriched around theheterochromatin regions of the centromeres, whichare largely shared with those found in ddm1 and fpgs1mutants. Given that reduced DNA methylation isfound only at certain sequence contexts, it is alsopossible that the inefficient histone methylationmight indirectly affect DNA methylation. For exam-ple, the CHG DNA methylation in the Pro35S-NPTIItransgene was only moderately reduced, while amore significant reduction in H3K9me2 was found inthe 35S promoter of the mat4 mutant. However, thishypothesis is hard to test as SAM is a commonsubstrate for both DNA and histone methylation.

Reduced SAMmust more or less affect both DNA andhistone methylation.

In humans, three MAT genes encode MATa1,MATa2, and MATb. MATa1 and MATa2 can formhomodimers or tetramers that have different affinity forsubstrates, and MATa2 can interact with MATb tostrengthen the activity of (MATb)4 (Murray et al., 2014).In Saccharomyces cerevisiae, when two MATs that share92% identity in amino acid sequence are disrupted, themutants display opposite phenotypes to the excessethionine added in the growth medium (Thomas andSurdin-Kerjan, 1987, 1991; Thomas et al., 1988), indi-cating that different MAT isoforms act on their ownrhythms. There are four close MAT homologs in Ara-bidopsis. However, we found that in the in vitro assaysMAT3 has the highest activity, while MAT1, MAT2,and MAT4 have comparable but lower activities.Among them, MAT4 is predominant as its missensemutation reduces SAM and DNA methylation togreater extent than MAT1 and MAT2 loss-of-functionmutations and can release the silencing of Pro35S::NPTII, while other mutations cannot. We used the

Figure 10. MAT4 interactswith differentMATs in plants. A,MAT4 interactedwithMAT1,MAT2,MAT3, orMAT4 itself in a proteinco-IP assay. Total proteins were extracted from Arabidopsis protoplasts transiently coexpressing the MAT4-FLAG with MAT1-,MAT2-, MAT3-, MAT4-GFP, or GFP (as a negative control) plasmids and immunoprecipitated with anti-GFP beads. The co-IPproteins were immunoblotted with anti-FLAG and anti-GFP antibodies. B, Protein pull-down assay for MAT4 interaction withMAT1, MAT2, MAT3, or MAT4 itself. Total proteins were isolated from E. coli coexpressing MAT4-His with GST-MAT1, -MAT2,-MAT3, -MAT4, or GST itself (as a negative control) and immunoprecipitated with Glutathione-Sepharose beads. The co-IPproteins were immunoblotted with anti-His and anti-GST antibodies. C, Gel filtration analyses. The 0.5 mg of total proteinsextracted from ;20 g of the 15-d-old seedlings of MAT4-FLAG was applied to an ANTI-FLAG M1 Agarose Affinity Gel. Theproteins were eluted using 0.5 mg/mL FLAG Peptide. The elution at the peaks was used for LC-MS analysis. D, LC-MS/MS analysesof the proteins of the three peaks in C. Cov indicates the percentage of sequence coverage (%); Seq (sig) indicates number ofsignificant sequences.





CRISPR/Cas9 technique, but failed to get the loss-of-function homozygous mutant of MAT4. These resultsindicate that MAT4 is an essential gene for plant growthand development in Arabidopsis. We found that theMAT4 promoter driving different MAT cDNAs cancomplement mat4 mutants. However, only a few mat4mutants can be complemented by the MAT1 promoterdriving MAT4 cDNA, which might be caused by highexpression of ProMAT1::MAT4-GFP in these transgeniclines, likely because the T-DNAs were inserted inenvironment-friendly sites in the genomic region. Nev-ertheless, mat4 mutants were not complemented inseveral ProMAT2::MAT4-GFP and ProMAT3::MAT4-GFPtransgenic lines. These results indicate the expressionpattern of MAT4, but not MAT4 protein itself, is impor-tant for its predominant biological roles in Arabidopsis.Using pull-down assays and co-IP assays, we found thatMATs interactedwith each other both in vitro and in vivo,suggesting that MATs can also form homo- and/orhetero-oligomers of different sizes in Arabidopsis. How-ever, more attempts or even crystallographic structuralanalyses should be carried out to obtainmore informationabout their precise composition in Arabidopsis.

MATERIALS AND METHODS

Plant Growth Conditions, Mutant Screening,and Identification

Arabidopsis (Arabidopsis thaliana) seeds were sterilized with 0.5% NaClOand then sown into Murashige and Skoog (MS) medium, which contained 2%(w/v) Suc and 0.8% (w/v) agar. After 3 d at 4°C, the plates were transferred togrowth chambers with long-day conditions (23 h of light/1 h of dark) at 22°C.Generally, 10-d-old seedlings were transferred to soil and cultured in a green-house with long-day conditions (16 h of light/8 h of dark) at 20°C.

We used L119, which harbors two transgenes, ProRD29A::LUC and Pro35S::NPTII, as the wild-type line. The mutants were selected from an EMS-mutagenized population of L119 for resistance to 25 mg/L Kan, whereas theL119 lines are typically sensitive under this condition. Map-based cloning wasconducted to identify the mutation. We crossed our mutants with Ler andobtained the F2 population, from which we selected Kan-resistant lines(519 total) for mapping.

Formutant complementation, the full genomic lengthofMAT4, including the2,221-bp promoter and the overall genomic sequence, was cloned intopCAMBIA1307. The construct in Agrobacterium tumefaciens strain GV3101 wastransformed into mat4 by the floral dip method (Clough and Bent, 1998). Thehomozygous transgenic lines were selected onMSmedium supplemented with30 mg/L hygromycin from the next T2 generation. All of the primers used inthis study are listed in the Supplemental Table S2.

RNA Analysis

For real-time RT-qPCR, total RNA was isolated using TRIzol reagent(Invitrogen) from 15-d-old seedlings, and 4 mg RNA was reverse-transcribedinto cDNA using the GOSCRIPT reverse transcription system (PromegaA5001). Then, 2mL of diluted (103) cDNAmixture was used as the template fora PCR assay using 20 mL of SYBR GreenMaster Mix (TaKaRa) performed on aStep One Plus system (Applied Biosystems). The experiments were per-formed in three independent biological replicates with technical triplicate. Allof the primers used in the real-time PCR assay are listed in SupplementalTable S2.

Subcellular Localization

The full-length cDNAofMAT4 fused with aGFP tag under the control of thesuper promoter was constructed in the pCAMBIA 1300 vector and the full

genomic length of MAT4, including the 2,221-bp promoter and the overall ge-nomic sequence, was cloned into pCAMBIA1300. The plasmids were extractedand purified with the Plasmid Maxprep Kit (VIGOROUS N001). Then, we in-troduced the plasmids into Arabidopsis protoplasts as previously described(Kong et al., 2015). After 14 to 16 h of incubation in light, the protoplasts wereviewed using a confocal microscope (Zeiss LSM 510META), and the GFP signalwas detected with 488-nm excitation. Empty GFP plasmids were used as acontrol. A. tumefaciens strain GV3101 carrying the same constructs were alsoinjected into Nicotiana benthamiana leaves. After 48 to 72 h of incubation, asection of the injected leaves was examined using the confocal microscope,and the GFP signal was detected using 488-nm excitation. An empty GFPconstruct was used as a control. We also obtained transgenic lines bytransforming this construct into L119 using A. tumefaciens strain GV3101.The 7-d growth of the T2 homozygous transgenic plants was used to detectfluorescence signals using a confocal microscope (Zeiss LSM 510 META)with 488-nm excitation.

Cellular Distribution of MAT4-FLAG

Next, 0.1 g of 15-d-old seedlings was ground into powder in liquid nitrogenand suspended with 200 mL isolation buffer (0.4 M Suc, 10 mM Tris-HCl, pH 8.0,10 mM MgCl2, 5 mM b-mercaptoethanol, and 1 mM PMSF), then filtered throughmicrocloth (Calbiochem 475855-1R), and the flow-through was centrifuged at2,800g for 10 min at 4°C. The supernatant was used for the cytosol, while theprecipitate was used for the nuclei after four washes with isolation buffer. Thenimmunoblotting using antibodies (H3,Millipore, 07-690; FLAG, Sigma-Aldrich,F3165; PEPC, Agrisera, AS09458) was carried out. Here, phosphoenolpyruvatecarboxylase (PEPC) was used as the cytosol marker, while H3 was used as thenucleus marker.

Bisulfite Sequencing

GenomicDNAwasextractedusing theDNeasyPlantMiniKit (Qiagen69104)from 15-d-old seedlings. The EZ Methylation-Gold Kit (Zymo Research D5005)was used to analyze DNA methylation. Five hundred nanograms of DNA wasadded to the reaction, and all steps followed the protocol supplied in the kit.Nearly 50 ng treated DNA was added to the PCR reaction using the specificprimers listed in Supplemental Table S2. The PCR products were introducedinto the pMD18-T simple vector (Takara 6011), and at least 15 clones were se-quenced for each sample.

Histone Extraction and Immunoblotting

Histone proteins were extracted from 15-d-old seedlings following theprotocol as previously described (Li et al., 2012). The antibodies used in im-munoblotting were H3 (Millipore; 07-690), H3K9me1 (Millipore; 17-680),H3K9me2 (Abcam; ab1220), H3K27me1 (Millipore; 07-448), and H3K4me3(Millipore; 07-473). H3 was used as the loading control. The experiments wereperformed in three independent biological replicates.

Histone Immunofluorescence Staining Assay

The assaymainly followed apreviously describedprocess (Soppe et al., 2002)with subtle modifications. The nuclei were isolated from 15-d-old seedlings.After resuspending with sorting buffer, the nuclei were dropped on the slides toair dry. The nuclei were then postfixed using 4% paraformaldehyde in PBS for20 min, washed four times with PBS, and closed with signal enhancer (CellSignaling; 11932) for 30min at room temperature. After washing four times, theplates were incubated with primary antibody for 2 h at 37°C or overnight at 4°Ccovered with Parafilm. Then, the plates were washed four times in vats filledwith PBST (PBS addedwith 0.1% Tween 20), each for 5min, and incubatedwithsecondary antibody in dark for 1.5 h at room temperature. Then, the plates werewashed four times in vats filledwith PBST. Eightmicroliters of DAPI (1mg/mL)was added onto the slides to counterstain the nuclei. The slides were coveredwith cover glasses. The signal was observed with a confocal microscope (Leicasp5) and collected under the emission wavelength of 405 and 561 nm for DAPIand Rhodamine.

The primary antibodies used in this assaywere H3K9me1 (Millipore; 17-680,1:50, rabbit), H3K9me2 (Abcam; ab1220, 1:100, mouse), H3K27me1 (Millipore;07-448, 1:100, rabbit), andH3K4me3 (Millipore; 07-473, 1:100, rabbit). Secondaryantibodies used in this assay were Rhodamine Red conjugate-Goat anti-mouse


Meng et al.







(Invitrogen R6393) and Rhodamine Red conjugate-Goat anti-rabbit (InvitrogenR6394).

Whole-Genome Bisulfite Sequencing and Analyses

Genomic DNA was extracted from 15-d-old seedlings using DNeasy PlantMini Kit (Qiagen 69104). Two micrograms of DNA was used for bisulfitetreatment and library construction, and MethylC-seq was carried out using theHiSeq 2000 (Illumina).

Raw data were obtained from the whole-genome bisulfite sequencing usingthe Illumina HiSeq platform. Clean data were generated by trimming adaptorbases and removing low-quality reads. For data analysis, paired-end clean readswere mapped to the reference genome sequence of the Arabidopsis genome(TAIR10) with Bismark (Krueger and Andrews, 2011). The DMRs were deter-mined and identified as previously published (Zhao et al., 2014).

For investigation of DMR enrichment, we followed the previously describedanalysiswith somemodifications (Zhao et al., 2014). TheDNAmethylation levelin genes without TEs, S-TEs (shorter than 0.5 kb), and L-TEs (longer than 4 kb)were calculated.

RNA-Seq and Analysis

Total RNA was extracted from 15-d-old seedlings using the RNeasy PlantMini Kit (Qiagen 74904). Two micrograms of RNA was used for library con-struction, each sample with two replicates. The transcriptome data set used inthis studywas obtained using the IlluminaHiSeq platform, and 125-bp trimmedpaired-end reads with high quality were generated. The trimmed reads weremapped to the reference genome sequence of the Arabidopsis genome (TAIR10)using bowtie2 (http://computing.bio.cam.ac.uk/local/doc/bowtie2.html)with default settings (Langmead et al., 2009). Differential gene expressionanalyses were performed using edgeR (http://bioinf.wehi.edu.au/edgeR/;Robinson et al., 2010). We selected genes with fold change . 2 and P , 0.0001compared to the wild type as differential expression genes and TEs. The dis-tribution of differentially expressed genes and TEs in the chromosomes wasplotted by circos (Krzywinski et al., 2009). The categories of up-regulated TEswere divided as previously described (Wang et al., 2015a).

ChIP Assays

Nuclei were isolated from 15-d-old seedlings and fixed with 1% formalde-hyde, following the protocol as described previously (Saleh et al., 2008). Thepure nuclei were resuspended with 300 mL of cold nuclei lysis buffer, then thegenomic DNA was sonicated into 250- to 500-bp fragments, and the superna-tant was diluted with ChIP dilution buffer. Twenty microliters of protein A/GMagnetic beads (Millipore; 16-663) was added for 90 min at 4°C to decreasenonspecific combination with gentle rotation. All steps that needed to collectbeads were carried out on a Magnetic rack on ice. The antibody H3K9me2(Abcam; ab1220) was added with gentle rotation over night at 4°C to allowcombination. The magnetic beads were washed five times: one time with lowsalt wash buffer, one time with high salt wash buffer, one time with LiCl washbuffer, and two times with TE buffer. Each wash was 5 min at 4°C with gentlerotation. The protein and DNA complex were eluted by elution buffer at 65°Cand then incubated at 65°C for at least 6 h or overnight to reverse cross-linking.Next, the RNAs were digested using RNase A at 37°C for 2 h, and then theproteinase K was added to digest the protein at 65°C at least for 6 h. TheQIAquick PCR purification kit (Qiagen; 28106) was used to obtain high qualityDNA, then the concentrations of the DNA measured with Qubit Fluorometer3.0 (Invitrogen; Q33216) were adjusted to 50 pg/mL. Finally, 1 mL DNA wasused as the template in 20 mL of SYBR Green Master mix (TaKaRa) on a StepOne Plus machine (Applied Biosystems). The experiments were performed inthree independent biological replicates.

Measurement of SAM Contents by LC-MS

Sixteen-day-old seedlings were used for the subsequent measurements. Theextraction and determination methods were followed as previously describedwith somemodifications to extraction (Nikiforova et al., 2005).We added 300mLmethanol (v/v: 80% and precooled at 220°C) and 100 mL precooled methanolwith 15 mg/mL DTT to 100 mg plant samples that had been ground intopowder in liquid nitrogen, vortexed for 1 min, extracted for 30 min on ice andthen centrifuged at 4°C for 10 min at 12,000g. We added 300 mL precooledisopropanol and 100mL precooledmethanol with 15mg/mLDTT to resuspend

the precipitation, extracted for 30 min on ice, and then centrifuged at 4°C for10 min at 12,000g. The two supernatants were combined, filtered, and thenanalyzed via LC-MS. The experiments were performed in three independentbiological replicates with technical triplicate.

Acquisition of Knockout Mutants Using the CRISPR/Cas9 Assay

The targets were selected according to the http://www.crisprscan.org/?page=sequence website. Two targets were chosen for each gene, and primerswere designed. Fragments were amplified by PCR using pCBC-DT1T2 as atemplate (Wang et al., 2015b). After PCR products were purified, they weredigested using the restriction enzyme BsaI and ligated using T4 ligase in onesystem for 5 h at 37°C, 5 min at 50°C, and 10 min at 80°C. They were thentransformed into the competent cell of JM109. After incubation, the correct clonewas identified. The construct in A. tumefaciens strain GV3101 was transformedinto L119 by the floral dipmethod (Clough and Bent, 1998). The transgenic lineswere selected on MS medium supplemented with 30 mg/L hygromycin fromthe T1 generation and sequenced to obtain knockout lines.

Determination of the Activities of MATs

The activities of MATs were determined by measuring the productionamount of SAM after reactions. The purified proteins of MAT1-His, MAT2-His,MAT3-His, MAT4-His, andMAT4 (D246N)-His in Escherichia coliwere desaltedusing a 10-kD Centrifugal Filter Unit (Millipore UFC501096). The reaction wascarried out in a 200 mL mixture that included 40 mg MAT, 10 mM ATP, 5 mM

Met, 0.1 M Tris-HCl (pH 8.0), 0.02 MMgCl2, and 0.2 MKCl at 37°C for 20min, andthen the reaction was terminated by adding 800 mL of 75% acetonitrile and 1.2%formic acid. The reaction solution was transferred to a 10-kD Centrifugal FilterUnit and centrifuged at full speed in 4°C for 10 min. The solution was used forLC-MS analysis. The experiments were performed in three independent bio-logical replicates with technical triplicate.

Pull-Down Assays

The full-length cDNA of MAT1, MAT2, MAT3, and MAT4 fused with Hisand GST tags were constructed in PET30a and pGEX-4T-1. The relevant plas-mids were cotransformed into the Rosetta (DE3) strain of E. coli. TheGlutathione-Sepharose beads were used to purify the proteins. Then the pro-teins were eluted from the Glutathione-Sepharose beads using 10 mM reducedGSH in 50 mM Tris-HCl. The cell lysates before addition of Glutathione-Sepharose beads were used as input to detect whether two proteins wereboth expressed. Then products were detected by immunoblot using the anti-bodies of His and GST.

Co-IP Assays in Arabidopsis Protoplasts

The full-length cDNAs ofMAT1,MAT2,MAT3, andMAT4 fusedwith FLAGand GFP tags under the control of the super promoter were constructed in thepCAMBIA 1300 vector. The plasmids were extracted and purified with thePlasmid Maxprep Kit (VIGOROUS N001). Then, the two relevant plasmidswere cointroduced into Arabidopsis protoplasts as previously described (Konget al., 2015). After 14 to 16 h of incubation in light, the protoplasts were collectedand total protein was extracted using the immunoprecipitation buffer (50 mM

Tris-HCl, pH 7.6, 5 mM MgCl2, 150 mM NaCl, 10% glycerol, 0.5% Nonidet P-40,1 mM DTT, 1 mM PMSF, and protease inhibitor cocktail, 1:100, 1 plate per mL[Roche]) for 30 min on ice. The protein solution was centrifuged at 12,000g for15 min at 4°C. Ten microliters of GFP-Trap_A (Chromotek; gta-20) was addedto the supernatant, then gently rotated for 2.5 h at 4°C to allow combination.The GFP-Trap_A was washed five times using the immunoprecipitation bufferand then the immunoprecipitated products were detected by immunoblot us-ing the antibodies of GFP and FLAG.

Gel Filtration Assay

MAT4-FLAG was used to prepare the protein for gel filtration. More than20 g of MAT4-FLAG seedlings grown on MS medium for 15 d were collectedand then ground to a powder in liquid nitrogen. The protein was extractedusing IP buffer (10mMHEPES, 1mMEDTA, pH 8.0, 100mMNaCl, 10% glycerol,0.5%Triton X-100, 1 mM DTT, 1 mM PMSF, and protease inhibitor cocktail,




http://computing.bio.cam.ac.uk/local/doc/bowtie2.html

http://bioinf.wehi.edu.au/edgeR/

http://www.crisprscan.org/?page=sequence

http://www.crisprscan.org/?page=sequence


1 plate per mL [Roche]) for 30min on ice. Then the protein solutionwas centrifugedat 12,000g for 15 min at 4°C. The supernatant was added to the ANTI-FLAG M1Agarose Affinity Gel (Sigma-Aldrich; A4596), then gently rotated for 2.5 h at 4°C toallow combination. The FLAG Agarose was washed five times using the IP buffer,and the protein was eluted using 0.5mg/mL FLAG Peptide (Sigma-Aldrich; F3290).We prepared 0.5 mg protein for gel filtration analysis. The effluent of the indicatedpeaks was sent for LC-MS analysis.

Accession Numbers

The gene accession numbers that were used in this study are as follows:AT3G17390 (MAT4/SAM3/MTO3), AT1G02500 (MAT1/SAM1), AT4G01850(MAT2/SAM2), AT2G36880 (MAT3), DDM1 (AT5G66750), AT3G18780 (AC-TIN), AT5G52310 (RD29A), AT2G17690 (SDC), AT4G03650 (AtGP1), andBD298459.1 (TSIs). RNA-seq, and BS-seq data were deposited in the NationalCenter for Biotechnology Information Gene Expression Omnibus databaseunder accession number GSE84014.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. T-DNA insertion positions in L119.

Supplemental Figure S2. Growth phenotypes of mat4 mutants and Kan-resistant phenotypes of two ddm1 alleles.

Supplemental Figure S3. Effects of mat4 on DNA methylation throughoutthe five chromosomes.

Supplemental Figure S4. Complementation of reduced histone modifica-tion in mat4 by MAT4-FLAG.

Supplemental Figure S5. Confirmation of RNA-seq data by RT-qPCR andassociation between DNA methylation and gene expression.

Supplemental Figure S6. T-DNA insertion positions, expression levels,and SAM contents in mat1, mat2, and mat3.

Supplemental Figure S7. Whole genomic DNA methylation changes inmat1, mat2, and mat3.

Supplemental Figure S8. Kanamycin sensitivity of mat1, mat2, and mat3CRISPR/Cas9 mutants in L119.

Supplemental Figure S9. Complementation of mat4 by MAT4 driven bythe native MAT1 promoter.

Supplemental Figure S10. Growth and development phenotypes of mat1-mat2 double mutants and embryogenic defects of mat2-c19mat4 doublemutants.

Supplemental Figure S11. The interaction of MAT1, MAT2, and MAT3with different MATs as determined by coimmunoprecipitation assays.

Supplemental Figure S12. The interaction of MAT1, MAT2, and MAT3with different MATs as determined by protein pull-down assays.

Supplemental Table S1. LC-MS/MS analyses of affinity copurified pro-teins from MAT4-FLAG seedlings.

Supplemental Table S2. Primers used in this study.

Supplemental Data Set S1. Hypo-differentially methylated regions of CGin mat4.

Supplemental Data Set S2. Hypo-differentially methylated regions ofCHG in mat4.

Supplemental Data Set S3. Hypo-differentially methylated regions ofCHH in mat4.

Supplemental Data Set S4. Differentially expressed genes up-regulated inmat4.

Supplemental Data Set S5. Differentially expressed genes down-regulatedin mat4.

Supplemental Data Set S6. Differentially expressed TEs up-regulated inmat4.

Supplemental Data Set S7. Differentially expressed TEs down-regulatedin mat4.

ACKNOWLEDGMENTS

We thank Dr. Zhen Li and Dr. Zhongzhou Chen of China AgriculturalUniversity for assistance with LC-MS analyses and gel filtration, respectively.

Received February 13, 2018; accepted March 16, 2018; published March 23,2018.

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METHIONINE ADENOSYLTRANSFERASE4 Mediates · MAT4 knockout mutations generated by CRISPR/Cas9 were lethal, indicating that MAT4 is an essential gene in Arabidopsis. MAT1, 2, and 4