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First published online June 15, 2007; 10.1104/pp.107.099531 Plant Physiology 144:1913-1923 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Mutations in the Type II Protein Arginine Methyltransferase AtPRMT5 Result in Pleiotropic Developmental Defects in Arabidopsis1,[C],[OA]College of Life Science and Technology, Shanxi University, Taiyuan 030006, China (Y.P.); State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Y.P., L.N., F.L., C.L., J.Z., X.K., X.C.); and Graduate School, Chinese Academy of Sciences, Beijing 100039, China (L.N., F.L., J.Z., X.K.)
Human PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) encodes a type II protein arginine (Arg) methyltransferase and its homologs in animals and yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe) are known to regulate RNA processing, signal transduction, and gene expression. However, PRMT5 homologs in higher plants have not yet been reported and the biological roles of these proteins in plant development remain elusive. Here, using conventional biochemical approaches, we purified a plant histone Arg methyltransferase from cauliflower (Brassica oleracea) that was nearly identical to AtPRMT5, an Arabidopsis (Arabidopsis thaliana) homolog of human PRMT5. AtPRMT5 methylated histone H4, H2A, and myelin basic protein in vitro. Western blot using symmetric dimethyl histone H4 Arg 3-specific antibody and thin-layer chromatography analysis demonstrated that AtPRMT5 is a type II enzyme. Mutations in AtPRMT5 caused pleiotropic developmental defects, including growth retardation, dark green and curled leaves, and FlOWERING LOCUS C (FLC)-dependent delayed flowering. Therefore, the type II protein Arg methyltransferase AtPRMT5 is involved in promotion of vegetative growth and FLC-dependent flowering time regulation in Arabidopsis.
Protein Arg methylation, a posttranslational modification event, plays essential roles in modulating transcription, RNA processing, DNA repair, and signal transduction (Bedford and Richard, 2005  ; Lee et al., 2005a). According to the biochemical characteristics, protein Arg methyltransferases (PRMTs) are classified into type I and type II enzymes. Type I enzymes form monomethyl-Arg (MMA) and asymmetric dimethyl-Arg (ADMA) residues, whereas type II enzymes catalyze the formation of MMA and symmetric dimethyl-Arg (SDMA) residues. To date, nine human PRMTs (PRMT1–PRMT9) have been identified. Among them, PRMT1, PRMT3, PRMT4/CARM1 (coactivator-associated Arg methyltransferase 1), PRMT6, and PRMT8 are type I enzymes (Bedford and Richard, 2005; Lee et al., 2005b), whereas PRMT5 (Branscombe et al., 2001), PRMT7 (Lee et al., 2005c), and PRMT9 (Cook et al., 2006) belong to the type II group. Human PRMT5 represents the major type II enzyme and methylates histone H2A, H3, H4, and nonhistone proteins, including myelin basic protein (MBP), small nuclear ribonucleoproteins (snRNPs; SmD1 and SmD3), methyl-DNA-binding domain proteins (MBDs), and transcription elongation factor SPT5 (suppressor of Ty 5) (Branscombe et al., 2001; Meister et al., 2001; Kwak et al., 2003; Pal et al., 2004; Ancelin et al., 2006; Tan and Nakielny, 2006). PRMT5 has been implicated in modulating RNA processing, signal transduction, and gene expression (Pollack et al., 1999; Branscombe et al., 2001; Friesen et al., 2001; Fabbrizio et al., 2002; Friesen et al., 2002; Meister and Fischer, 2002; Pal et al., 2004; Ancelin et al., 2006). Unlike PRMT1 and PRMT4, two important type I PRMTs that are associated with gene activation, PRMT5 usually functions in repressing target gene expression (Fabbrizio et al., 2002; Pal et al., 2004; Ancelin et al., 2006).
PRMT5 homologs have been identified in yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe), fruitfly (Drosophila melanogaster), mouse, and filamentous fungus (Gilbreth et al., 1996 However, the importance of protein Arg methylation in Arabidopsis (Arabidopsis thaliana) has just emerged and the roles of the PRMT5 counterpart in Arabidopsis remain unclear. Using a biochemical approach, we have previously purified a PLANT HISTONE ARGININE METHYLTRANSFERASE10 (PHRMT10) from cauliflower (Brassica oleracea; L.F. Niu, F.L. Lu, Y.X. Pei, C.Y. Liu, and X.F. Cao, unpublished data). Simultaneously, we successfully purified another histone H4 Arg methyltransferase, PHRMT5, from cauliflower. PHRMT5 and its Arabidopsis homolog AtPRMT5 symmetrically methylate histone H4 at Arg-3. Loss-of-function mutations in AtPRMT5 cause pleiotropic phenotypes. Most interestingly, AtPRMT5 is found to play important roles in promoting seedling growth and the transition from vegetative to reproductive development. These data provide the experimental evidence supporting that the type II PRMT AtPRMT5 is involved in regulating plant vegetative growth and control of flowering time in Arabidopsis.
Purification and Characterization of a Histone H4 Methyltransferase By following histone methyltransferase activity, we have previously purified a histone H4 Arg 3 (H4R3) methyltransferase, PHRMT10, from cauliflower (L.F. Niu, F.L. Lu, Y.X. Pei, C.Y. Liu, and X.F. Cao, unpublished data). During the purification procedure, multiple histone methyltransferase activity peaks were observed. The strongest activity against histone H4 was in peak b (Fig. 1A ; data not shown). This activity peak was pooled and further fractionated on a Superdex200 gel filtration column. Silver staining of an SDS-PAGE gel containing the column fractions revealed that a polypeptide band migrating at around 67 kD coeluted with the histone methyltransferase activity (Fig. 1B). This band was excised and subjected to mass spectrometry analysis. Peptides derived from liquid chromatography-tandem mass spectrometry (MSMS) showed the highest similarity to the Arabidopsis PRMT (AtPRMT) encoded by the gene At4g31120 (Fig. 1, C and D). We named this protein PHRMT5 for PLANT HISTONE ARGININE METHYLTRANSFERASE5. Mass spectrometry analysis of histone H4 methylated by PHRMT5 revealed that methylation occurred at histone H4R3 in both mono- and dimethylated manners based on the peptide corresponding to the first five amino acids of histone H4 (Fig. 1E). Compared with human PRMT proteins, AtPRMT coded by At4g31120 showed the highest similarity (47% identical at the amino acid level) to human PRMT5 and we named it AtPRMT5. AtPRMT5 contains 23 exons and encodes a 642-residue protein with conserved catalytic core including methyltransferase region I, posts I, II, III, and THW loop of the PRMT family (Fig. 2 ).
To substantiate the methyltransferase activity of AtPRMT5, recombinant glutathione S-transferase (GST)-AtPRMT5 fusion protein was analyzed. Figure 3A showed both Coomassie blue-stained gels (a and c) and autoradiographs (b and d) of the proteins methylated in the reactions. Among the four core histones (H2A, H2B, H3, and H4) assayed, H4 was preferentially methylated and H2A was also methylated in vitro (Fig. 3A, b). In addition to histones, nonhistone protein MBP was also the substrate of AtPRMT5 (Fig. 3A, d). PHRMT5 mono- and dimethylated histone H4R3 and PRMT5 in animals could symmetrically dimethylate histone H4R3. Therefore, to determine whether AtPRMT5 also showed the same type II methyltransferase specificity, western blotting was performed and calf thymus histone methylated by AtPRMT5 was recognized by the antibody against symmetric dimethyl histone H4R3 (Fig. 3B), as was calf thymus histone methylated by PHRMT5 (Fig. 3C). To further confirm that AtPRMT5 was a type II methyltransferase, a thin-layer chromatography (TLC) assay was performed after acidic hydrolysis of histone H4 that was methylated by AtPRMT5 in the presence of 3H-labeled S-adenosyl-L-Met (SAM). Compared with MMA and either ADMA or SDMA standards, we observed that the 3H-labeled methylated Args comigrated with both MMA and SDMA, but not ADMA (Fig. 3D). Hence, the mass spectrometry analysis, western blot, and TLC analysis support the conclusion that both AtPRMT5 and PHRMT5 mono- and symmetrically dimethylate histone H4 at R3 and that AtPRMT5 is a bona fide type II PRMT.
Isolation and Identification of atprmt5 Mutants To investigate the functions of AtPRMT5 in growth and development in Arabidopsis, we searched for atprmt5 T-DNA insertion mutant alleles and obtained atprmt5-1 and atprmt5-2. The atprmt5-1 mutant contains two inverted T-DNA insertions in the 21st exon, while atprmt5-2 has an insertion in the 22nd exon (Fig. 4A ). Each insertion was confirmed by PCR and sequencing analysis. In contrast to the RNA-blot analysis in which neither full-length nor truncated form of AtPRMT5 transcript was detectable in atprmt5 mutants (Fig. 4B, top sections), low levels of an N-terminally truncated form of AtPRMT5 were detected (Fig. 4B, bottom sections), indicating that atprmt5-1 and atprmt5-2 might not be null alleles.
Lesions in AtPRMT5 Cause Pleiotropic Developmental Defects in Arabidopsis Compared with the wild-type accession Columbia (Col), atprmt5 mutants showed pleiotropic phenotypes. The atprmt5 mutants displayed growth retardation such that both the size of cotyledons and rosette leaves and the length of primary roots in atprmt5-1 and atprmt5-2 mutants were strikingly smaller and shorter than those of wild-type Col during early vegetative development (Fig. 4, C and D). The leaves of atprmt5 mutants continued to expand and eventually reached the same size as Col leaves. In addition to growth retardation, some of the early juvenile leaves were curled in atprmt5 mutants as compared with wild-type Col (Fig. 4E). At later developmental stages, when the leaves of wild-type Col had senesced, atprmt5 leaves were more curled and dark green in color. Most obviously, mutations in AtPRMT5 led to dramatically delayed flowering time and increased number of rosette and cauline leaves, indicating that AtPRMT5 was involved in regulating the transition from vegetative to reproductive development (Fig. 4F). To characterize the atprmt5 mutants genetically, F1 progenies from crosses between wild-type Col and atprmt5 mutants were generated. All F1 plants displayed normal developmental phenotypes. In the F2 generation, plants exhibited a 1:3 segregation ratio of mutant to wild-type phenotypes and all plants showing the mutant phenotypes were atprmt5 homozygous. These genetic analyses revealed that atprmt5 mutants were recessive and controlled by a single locus. F1 progenies (atpmrmt5-1 x atprmt5-2) from reciprocal crosses between atprmt5-1 and atprmt5-2 displayed the same phenotype as their parents at both early and late developmental stages (Fig. 4F; data not shown), indicating that atprmt5-1and atprmt5-2 mutants are allelic. An extra copy of genomic DNA from AtPRMT5 rescued atprmt5 mutant phenotypes, indicating that the above pleiotropic phenotypes indeed were caused by disruption of AtPRMT5 (data not shown).
The floral transition is one of the most important plant developmental processes that influence reproductive success. In Arabidopsis, flowering time is regulated by four major pathways, including photoperiod, gibberellin (GA), vernalization, and autonomous pathways (Simpson et al., 1999
All above physiological characters of atprmt5 mutants were similar to those late-flowering mutants in the autonomous pathway. Autonomous pathway genes control flowering time mainly through repressing the major flowering repressor FLOWERING LOCUS C (FLC), a MADS-box transcription factor that quantitatively blocks the floral transition in Arabidopsis (Michaels and Amasino, 1999 ; Sheldon et al., 1999, 2000b, 2002, 2006; Rouse et al., 2002; Liu et al., 2004; Jean Finnegan et al., 2005). Therefore, to investigate whether expression of FLC was controlled by AtPRMT5, we examined mRNA levels of FLC in wild-type Col and atprmt5 plants. Expression of FLC was dramatically increased due to mutations in AtPRMT5 (Fig. 5B, top section). As a result, expression of SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1)/AGAMOUS-LIKE20, a floral integrator downstream of FLC, was greatly reduced (Fig. 5B, middle section). We also generated double mutants between atprmt5 and flc-3, a null allele mutant of FLC (Michaels and Amasino, 1999). Homozygous progeny of atprmt5/flc double mutants flowered significantly earlier than atprmt5 (Fig. 5C, a and b). Taken together, we concluded that AtPRMT5 regulates flowering time through the autonomous pathway and the late-flowering phenotype of atprmt5 is FLC dependent.
In humans, PRMT5 has been implicated in transcriptional repression of the cyclin E promoter by symmetric dimethylation of histone H4R3 (Fabbrizio et al., 2002 In addition to FLC, it was possible that upstream regulators of FLC were affected by AtPRMT5. To test this possibility, real-time PCR was performed to evaluate transcript levels of FLC modulators. Among eight repressors and 16 activators of FLC, only the mRNA level of FLOWERING LOCUS K (FLK), a component of autonomous pathway, was increased by 2-fold, and ARP6 (ACTIN-RELATED PROTEIN6), an activator of FLC, was decreased by 2-fold in atprmt5 mutants as compared to wild-type Col (Fig. 6 ).
In this study, we purified PHRMT5 from cauliflower, a histone H4 methyltransferase that confers the strongest histone H4 methyltransferase activity. Peptide sequences obtained by mass spectrometry showed that PHRMT5 is an ortholog of Arabidopsis AtPRMT5. In vitro methyltransferase activity assays showed that recombinant GST-AtPRMT5 methylates histone H4, H2A, and MBP, similar to PRMT5 in animals. In addition, recombinant AtPRMT5 and purified PHRMT5 catalyze symmetric dimethylation at histone H4R3, indicating that AtPRMT5 and PHRMT5 are type II PRMTs. Mutations in AtPRMT5 cause pleiotropic developmental defects, including seedlings growth retardation, darker green color, curled leaves, and delayed flowering, which indicates that AtPRMT5 may be involved in regulating fundamental cellular processes and development in Arabidopsis.
At the young juvenile stage, a striking defect of atprmt5 plants is the growth retardation, which indicates that wild-type AtPRMT5 enhances vegetative development. Consistent with this phenotype, yeast cells carrying the mutation in Skb1 are less elongated in morphology and show a slower rate of cell growth (Gilbreth et al., 1996
The regulation of flowering time by developmental signals and external environmental clues is a complex biological process and can be regulated at transcriptional and posttranscriptional levels (Henderson and Dean, 2004
Although FLC mRNA levels were up-regulated in atprmt5 plants, we did not observe any significant difference in histone modifications between wild-type and atprmt5 plants at FLC locus under our conditions (12-d-old seedlings grown at 23°C under LD condition; Fig. 5, B and D). A similar situation was observed in loss of function of a histone acetyltransferase (Deng et al., 2007
Purification of the Histone H4 Methyltransferase
One kilogram upper layer of cauliflower (Brassica oleracea) was homogenized in a waring blender in liquid nitrogen and further in 1 L buffer A [50 mM Tris-HCl, 0.1 mM EDTA, 20 mM (NH4)2SO4, 25% (v/v) glycerol, 2 mM dithiothreitol (DTT), and 0.2 mM phenylmethylsulfonyl fluoride (PMSF), pH 8.0] supplemented with protease inhibitor cocktail (Roche) for three times of 10 s bursts (Chamovitz et al., 1996
To identify the 65 kD band that coeluted with H4 histone methyltransferase, the Coomassie-stained 65 kD band was excised, digested with trypsin, and subjected to liquid chromatography-MSMS analysis according to standard procedure. DTA files for MSMS spectra were generated by Bioworks software (ThermoFinnigan) and searched against Arabidopsis (Arabidopsis thaliana) database using the Global Proteome Machine search engine (Craig et al., 2004
Full-length AtPRMT5 coding sequence was amplified from cDNA made from the inflorescence of wild-type Col Arabidopsis. Primers used for AtPRMT5 cloning were cx1108 (5'-CATGCCATGGATATGCCGCTCGGAGAGAGAGGAGGATGGG-3') and cx0927 (5'-TCCCCCGGGAAAAGTCTCTAATCCTAAAGGC-3'). Recombinant GST-AtPRMT5 was expressed in Escherichia coli strain BL21 (RIL) and purified using glutathione Sepharose beads (Amersham Biosciences). Purified recombinant protein or column fractions were incubated with indicated substrates in the presence of the methyl donor, S-adenosyl-L-[methyl-3H]Met (Amersham Biosciences), in HMT buffer (20 mM Tris-HCl, pH 8.0, 4 mM EDTA, 1 mM PMSF, 0.5 mM DTT) for 1 to 3 h at 30°C. Proteins were separated by 15% SDS-PAGE and visualized by Coomassie staining. Gels were then treated with Amplifier (Amersham Biosciences) for 15 to 30 min, dried, and exposed to Kodak Biomax MS film at –80°C for the appropriate time.
Proteins were separated by 10% or 15% SDS-PAGE and transferred to Hybond ECL membrane (Amersham Biosciences) or Immobilon P SQ polyvinylidene difluoride membrane (Millipore) for histones. Supersignal West Dura substrate (Pierce) was used to detect the HRP-conjugated secondary antibodies. Antibodies used for western blot are antisymmetric dimethyl-H4R3 (H3R3 SDM; Abcam, ab5823) and anti-histone H4 (Upstate, 07-108).
In vitro histone methylation reactions (50 µL) were performed with 3H-labeled SAM (Amersham biosciences), calf thymus histone H4 (Roche), and appropriate enzymes. Methylation reaction mixtures were hydrolyzed in ampule tubes by adding 5 volumes 6 N HCl (250 µL). The tubes were heat sealed and incubated at 110°C for 24 h. The reaction mixtures were then dried completely on a heat block set at 90°C. Twenty microliters of water was added into each tube and the tubes were let stand for about 30 min. Twenty-five percent hydrolyzed amino acids (5 µL) were mixed with 30 nmol Arg standards (either SDMA plus MMA or ADMA plus MMA) and spotted onto TLC plate (silica gel 60 F254 with concentrating zone, Merck). The TLC plate was run with ammonium hydroxide:chloroform:methanol:water (2:0.5:4.5:1) as previously reported (Friesen et al., 2001
All the Arabidopsis lines used in this study were the Col ecotype. flc-3 and fld-4 seeds are the kind gifts of Dr. R. Amasino. For flowering time measurements, plants were grown side by side in Versatile Environmental test chamber (MLR-350H, SANYO) at 23°C under 16 h of light followed by 8 h of darkness for LD or 8 h of light followed by 16 h of darkness for SD. For vernalization treatment, atprmt5 mutants and wild-type plants were grown in soil side by side at 2°C to 6°C for 6 weeks under SD before transferred to 23°C under LD condition. Nonvernalized control plants were grown under SD at 2°C to 6°C for 3 d before being transferred to 23°C under LD condition. For GA treatment, 20 µM GA3 solution was sprayed twice a week under SD until flowering. GA3 was dissolved in ethanol, therefore, equal amounts of ethanol was sprayed as untreated control. Flowering time was measured by counting the total numbers of rosette and cauline leaves at flowering.
atprmt5-1 (SALK_065814) and atprmt5-2 (SALK_095085) mutants were obtained from the SALK collection (http://signal.salk.edu/). T-DNA border primer cx101 LBb1 (5'-GCGTGGACCGCTTGCTGCAACT-3') or cx102 LBa1 (5'-TGGTTCACGTAGTGGGCCATCG-3') was used to amplify DNA from mutants. Gene-specific primers were cx166 (5'-TCTTGTGACAAAAATACAGCACAA-3') and cx167 (5'-CATCCATTTGGCAGGTTAAGGC-3') for atprmt5-1 and cx168 (5'-ATGTTTTGTCAAACGGCCGAG-3') and cx169 (5'-TCTACCAAGCGATGCTGGCTC-3') for atprmt5-2. The flc-3 seed was a gift from Dr. R. Amasino. Primers used for flc-3 genotyping were cx743 (5'-TTGCATCACTCTCGTTTACCC-3') and cx744 (5'-GCGTCACAGAGAACAGAAAGC-3'). T-DNA insertion fragments from PCR were further confirmed by sequencing.
mRNA expression levels were measured either by real-time PCR or by RNA gel blot. Seedlings with four to five rosette leaves of Col and atprmt5 plants grown in Murashige and Skoog plates with 3% Suc were used to extract total RNA (Liu et al., 2005
Twelve- or 14-d-old plants with four to five true leaves were harvested from Murashige and Skoog plates containing 3% Suc grown under LD at 23°C. ChIP assay was performed as previously described (Johnson et al., 2002 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AtPRMT5: NP_194841; hPRMT5: NP_006100; mPRMT5: NP_038796; SpSkb1: P78963; and ScHs17: NP_009691.
Wang et al. (Wang X, Zhang Y, Ma Q, Zhang Z, Xue Y, Bao S, Chong K [2007] SKB1-mediated symmetric dimethylation of histone H4R3 controls flowering time in Arabidopsis. EMBO J 26: 1934–1941) have also found that SKB1-mediated symmetric dimethylation of histone H4R3 controls flowering time in Arabidopsis.
We thank Dr. Haiyan Zheng (Robert Wood Johnson Medical School, Piscataway, NJ) for conducting the mass spectrometric analysis, Dr. R. Amasino (University of Wisconsin, Madison, WI) for providing flc-3 and fld-4 seeds, Dr. Ian Henderson (University of California, Los Angeles) for English editing, and the Arabidopsis Biological Resources Center at Ohio State for providing SALK T-DNA-insertion lines. Received March 14, 2007; accepted June 8, 2007; published June 15, 2007.
1 This work was supported by the National Basic Research Program of China (grant no. 2005CB522400 to X.C.), by the National Natural Science Foundation of China (grant nos. 30430410, 30621001, and 30325015 to X.C. and 30571032 to C.L.), and by the Chinese Academy of Sciences (grant no. CXTD–S2005–2). 
2 These authors contributed equally to the article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Xiaofeng Cao (xfcao{at}genetics.ac.cn).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.099531 * Corresponding author; e-mail xfcao{at}genetics.ac.cn; fax 86–10–64873428.
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TOP
ABSTRACT
RESULTS
gene is used as a control for constitutive expression. Bottom sections: RT-PCR analyses were performed for the N-terminally expressed AtPRMT5 (AtPRMT5N) in wild-type Col and atprmt5-1 and atprmt5-2 mutants. Equal amounts of cDNAs were determined by RT-PCR at the Actin locus. C to F, Pleiotropic phenotypes of atprmt5 mutants. Growth retardation of young seedling leaves at 12 d (C) and primary roots at 9 d (D). E, Comparison of rosette leaves between wild-type Col and atprmt5 plants. F, atprmt5 mutants are late flowering. Plants shown here are 8 weeks old grown at 23°C under LD. aprmt5-1 x aprmt5-2 means F1 progeny from the crosses between aprmt5-1 and aprmt5-2.
and 
forms are detected.
