MEA proteins are required for the repression of the maternal allele of <i>UCL1</i>.

<p>(A) Location of qRT-PCR primer sets used to detect the expression of <i>UCL1</i>. (B) Comparison of the expression levels of <i>UCL1</i> in wild-type L<i>er</i> and <i>mea-3</i> endosperm at 3 DAP. The expression of <i>UCL1</i> in the <i>mea-3</i>/<i>mea-3</i> mutant was set to 1 and the error bar represents the standard deviation of three independent samples. (C) Analysis of the allele-specific expression of <i>UCL1</i> using a CAPS marker. RT-PCR analysis was performed on RNA isolated from the endosperms of RLD females crossed with L<i>er</i> males, L<i>er</i> females crossed with RLD males, and <i>mea-3</i> or <i>fie-1</i> females (L<i>er</i> background) crossed with RLD males. These products were digested with <i>Eco</i>RI. The L<i>er</i> allele shows a 276 bp band, whereas the RLD allele was cut into a 222-bp band after <i>Eco</i>RI digestion. (D) Sequencing chromatograms of the RT-PCR products of <i>UCL1</i> at the distinguished SNP regions showing allele-specific expression. RNAs were isolated from endosperms resulting from reciprocal crosses between RLD and L<i>er</i> ecotypes and in crosses between the female <i>mea-3</i> or <i>fie-1</i> mutant and the male RLD.</p

Control of Paternally Expressed Imprinted <i>UPWARD CURLY LEAF1</i>, a Gene Encoding an F-Box Protein That Regulates CURLY LEAF Polycomb Protein, in the <i>Arabidopsis</i> Endosperm

<div><p>Genomic imprinting, an epigenetic process in mammals and flowering plants, refers to the differential expression of alleles of the same genes in a parent-of-origin-specific manner. In <i>Arabidopsis</i>, imprinting occurs primarily in the endosperm, which nourishes the developing embryo. Recent high-throughput sequencing analyses revealed that more than 200 loci are imprinted in <i>Arabidopsis</i>; however, only a few of these imprinted genes and their imprinting mechanisms have been examined in detail. Whereas most imprinted loci characterized to date are maternally expressed imprinted genes (MEGs), <i>PHERES1</i> (<i>PHE1</i>) and <i>ADMETOS</i> (<i>ADM</i>) are paternally expressed imprinted genes (PEGs). Here, we report that <i>UPWARD CURLY LEAF1</i> (<i>UCL1</i>), a gene encoding an E3 ligase that degrades the CURLY LEAF (CLF) polycomb protein, is a PEG. After fertilization, paternally inherited <i>UCL1</i> is expressed in the endosperm, but not in the embryo. The expression pattern of a <i>β-glucuronidase</i> (<i>GUS</i>) reporter gene driven by the <i>UCL1</i> promoter suggests that the imprinting control region (ICR) of <i>UCL1</i> is adjacent to a transposable element in the <i>UCL1</i> 5′-upstream region. Polycomb Repressive Complex 2 (PRC2) silences the maternal <i>UCL1</i> allele in the central cell prior to fertilization and in the endosperm after fertilization. The <i>UCL1</i> imprinting pattern was not affected in paternal PRC2 mutants. We found unexpectedly that the maternal <i>UCL1</i> allele is reactivated in the endosperm of <i>Arabidopsis</i> lines with mutations in cytosine DNA <i>METHYLTRANSFERASE 1</i> (<i>MET1</i>) or the DNA glycosylase <i>DEMETER</i> (<i>DME</i>), which antagonistically regulate CpG methylation of DNA. By contrast, maternal <i>UCL1</i> silencing was not altered in mutants with defects in non-CpG methylation. Thus, silencing of the maternal <i>UCL1</i> allele is regulated by both MET1 and DME as well as by PRC2, suggesting that divergent mechanisms for the regulation of PEGs evolved in <i>Arabidopsis</i>.</p></div

<i>UCL1</i> is paternally expressed in the endosperm.

<p>(A-C) Ovule and seeds derived from reciprocal crosses between the <i>UCL1_4</i>.<i>1k</i>::<i>GUS</i> transgenic plant and Col-0 wild type. (A) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>GUS</i> transgene in a wild-type ovule after emasculation. B) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>GUS</i> transgene in a wild-type seed at 1 day after pollination (DAP). (C) Expression of the paternally derived <i>UCL1_4</i>.<i>1k</i>::<i>GUS</i> transgene in the wild-type seed at 1 DAP. (D-F) Ovules and seeds resulting from reciprocal crosses between a <i>UCL1_4</i>.<i>1k</i>::<i>UCL1</i>:<i>GUS</i> transgenic plant and the wild type. (D) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>UCL1</i>:<i>GUS</i> transgene in a wild-type ovule after emasculation. (E) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>UCL1</i>:<i>GUS</i> transgene in a wild-type seed at 1 DAP. (F) Expression of the paternally derived <i>UCL1_4</i>.<i>1k</i>::<i>GUS</i> transgene in a wild-type seed at 1 DAP. Scale bars: 20 μm. (G) Sequencing chromatograms of RT-PCR products of <i>UCL1</i> showing allele-specific expression at a polymorphic site indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117431#pone.0117431.s001" target="_blank">S1 Fig</a>. Endosperm RNA was prepared in samples derived from reciprocal crosses between Col-0 and RLD ecotypes.</p

The ICR of <i>UCL1</i> is located between two LINE/L1 TEs.

<p>In the diagram of the promoter region of <i>UCL1</i>, the blue and red boxes indicate the distinct TEs in the <i>ATLINE1_1</i> family of the LINE/L1 superfamily in <i>At1065750</i>. The numbers to the left of the lines indicate the size of the promoters (in bp) fused to the <i>GUS</i> transgene. Transgenic plants carrying the <i>GUS</i> transgene fused to various lengths of the <i>UCL1</i> promoter were generated and the expression of the maternally derived transgenes was analyzed in seeds at 1 DAP, similarly as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117431#pone.0117431.g002" target="_blank">Fig. 2</a>.</p

DNA methylation is also relevant to maternal <i>UCL1</i> silencing.

<p>(A-D) Ovules after emasculation. (A) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>GUS</i> transgene in a wild-type ovule. (B) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>GUS</i> transgene in a <i>fie-1</i> mutant ovule. (C) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>GUS</i> transgene in a <i>dme-2</i> mutant female gametophyte. (D) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>GUS</i> transgene in a <i>met1–6</i> mutant ovule. (E-L) Seeds from plants hemizygous for the <i>GUS</i> transgene and heterozygous for <i>fie-1</i>, <i>dme-2</i>, or <i>met1–6</i>. The <i>fie-1</i>, <i>dme-2</i>, and <i>met1–6</i> mutants were used as females in crosses with wild-type pollen to characterize the expression of the <i>UCL1</i> maternal allele. (E) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>GUS</i> transgene in a wild-type seed at 1 DAP. (F) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>GUS</i> transgene in a <i>fie-1</i> mutant seed at 1 DAP. (G) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>GUS</i> transgene in a <i>dme-2</i> mutant seed at 1 DAP. (H) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>GUS</i> transgene in a <i>met1–6</i> mutant seed at 1 DAP. (I) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>UCL1</i>:<i>GUS</i> transgene in a wild-type seed at 1 DAP. (J) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>UCL1</i>:<i>GUS</i> transgene in a <i>fie-1</i> mutant seed at 1 DAP. (K) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>UCL1</i>:<i>GUS</i> transgene in a <i>dme-2</i> mutant seed at 1 DAP. (L) Expression of the maternally derived <i>UCL1_4</i>.<i>1k</i>::<i>UCL1</i>:<i>GUS</i> transgene in a <i>met1–6</i> mutant seed at 1 DAP. Scale bars: 50 μm. (M) Analysis of the allele-specific expression of <i>UCL1</i> using a CAPS marker. Endosperm RNAs were prepared from the female <i>met1–6</i> or <i>dme-2</i> mutant (Col background) crossed with the male RLD plant at both 3 DAP and 4 DAP. The RT-PCR products were analyzed before and after <i>Eco</i>RI digestion.</p

CpG methylation patterns of the 5′ upstream region of <i>UCL1</i> in the endosperm and embryo.

<p>Only CpG sites with fractional CpG methylation that is significantly different between the embryo and endosperm, and between the wild-type endosperm and <i>dme-2</i> endosperm are shown. Numbers on the x-axis represent CpG site positions (in bp) relative to the <i>UCL1</i> translational start site.</p

Proposed model for transcription repression by AtBBD1.

<p>In the absence of signal, AtBBD1 represses <i>AtJMT</i> gene expression by recruiting corepressor or HDAc through AtJAZ. In the presence of signal, JA-Ile is released and the SCF^COI1 complex degrades JAZ proteins. A putative activator (+) that binds to the JARE competes with AtBBD1 (repressor). In knockout plants, the putative activator dominantly occupies the JARE and <i>AtJMT</i> gene expression is activated higher than wild type. In the AtBBD1-overexpressing plant, AtBBD1(repressor) competes with the putative activator and dominantly occupies the JARE; therefore, <i>AtJMT</i> gene expression is repressed more than in wild type. Size of each circle represents relative abundance.</p

Structure of the <i>UCL1</i> locus in different <i>Arabidopsis</i> ecotypes.

<p>(A) Overview of the <i>UCL1</i> locus in different <i>Arabidopsis</i> ecotypes. The blue and red boxes indicate distinct TEs in the <i>ATLINE1_1</i> family of the LINE/L1 superfamily in <i>At1065750</i>. L<i>er</i>, RLD, and C24 do not include the long <i>ATLINE1_1</i> TE, whereas Col-0 and En-2 do. The numbers are in base pairs (bp) from the translation start site of <i>UCL1</i>. (B) Expression of the maternally derived <i>UCL1_2</i>.<i>7k</i>::<i>GUS</i> transgene in a wild-type seed at 1 DAP. (C) Expression of the maternally derived <i>UCL1_1</i>.<i>5k</i>::<i>GUS</i> transgene in a wild-type seed at 1 DAP. (D) Expression of the maternally derived <i>UCL1_2</i>.<i>7k</i>::<i>UCL1</i>:<i>GUS</i> transgene in a wild-type seed at 1 DAP. (E) Expression of the maternally derived <i>UCL1_1</i>.<i>5k</i>::<i>UCL1</i>:<i>GUS</i> transgene in a wild-type seed at 1 DAP. Scale bars: 50 μm.</p

Summary of the Y2H assay of OsJAZs.

1<p>The strength of each interaction was rated as strong (+++), medium (++), weak (+) or undetectable (−), as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052802#pone.0052802.s004" target="_blank">Figure S4</a>.</p>2<p>OsCOI1a(N475Y) is a point mutant in which asparagine at 475 has been changed to tyrosine.</p>3<p>OsCOI2(H391Y) is a point mutant in which histidine at 391 has been changed to tyrosine.</p>4<p>OsCOI2(F91Y) is a point mutant in which phenylalanine at 91 has been changed to tyrosine.</p>5<p>OsCOI2(N477Y) is a point mutant in which asparagine at 477 has been changed to tyrosine.</p>6<p>OsCOI2(F91Y, H391Y, N477Y) is a point mutant in which each amino acid at there position has been changed to tyrosine.</p>7<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052802#pone.0052802-Seo1" target="_blank">[21]</a>.</p

Identification of sequence element in the <i>BcNTR1</i> promoter region to which AtBBD1 binds.

<p>(A) Structures of reporter and activator genes used in Y1H assays. The promoter region of <i>BcNTR1</i>, −3518 to −3390, was divided into 3 segments and each segment was used as bait for Y1H assays. The control does not contain any of those segments. AtBBD1 was fused with the GAL4 activating domain (AD) as an activator. The position of the putative JARE is shown (▾). (B) The segment a was divided further into 8 subsegments (6 nt each) and each subsegment, a1 to a8, was mutated into 6 adenines. Each mutant segment was tested as bait in Y1H assays. (C) Subsegments a6 and a7 to which AtBBD1 bound, were dissected further by mutation in overlapping frames. In each mutant, 6 nucleotides were mutated into 6 adenines. Each mutant subsegment, M0–M5, was tested by Y1H assays. The sequence motif to which AtBBD1 binds is shown in bold. (D) Mutation analysis of the AtBBD1 binding element. Mutant series (CM1 to CMR) of JARE was created by changing a single nucleotide from purine to pyrimidine, or <i>vice versa</i>, in the fragment −2305 to −2278 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055482#pone-0055482-g004" target="_blank">Fig. 4A</a> as a bait and Y1H assays were carried out with AD-AtBBD1. CMR is a JARE in reverse orientation_.</p