Model illustrating the essential role of histone modifications during seed-to-seedling and vegetative-to-generative phase transitions.

<p>Repressive H3K27me3 marks on genes encoding key regulators of dormancy, such as <i>ABI3</i>, are deposited through the PRC2 complex, and replace the activating H3K4me3 mark in response to the environmental cue of moist chilling, similar to the vernalization response on the FLC locus. In order to fulfill its role as a flowering repressor, the FLC locus has to be re-activated after seed germination. ATXR7 might be involved in this process <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051532#pone.0051532-Tamada1" target="_blank">[46]</a>.</p

Expression analyses of histone methyltransferases (HMTs) in Arabidopsis Cvi seeds.

<p>(<b>A</b>) Class I SET-domain HMTs. (<b>B, C</b>) Class III SET-domain HMTs. Mean of three biological replicates +/− SE are shown. Note that the Y-axis for the RNA data is in log 10-scale.</p

Expression analyses and histone H3 methylation pattern changes of <i>CnABI3</i> in yellow-cedar.

<p>(<b>A</b>) Embryos (<b>B</b>) Megagametophytes. nChIP/qPCR (left column) and expression analyses (right column). Averages of three biological replicates are shown +/− SE. Mature yellow-cedar seeds were subjected to a full (12-week) dormancy-breaking treatment, or the full dormancy-breaking treatment followed by 1 day in germination conditions (“germinating”). Seedlings were at an early stage (4 mm radicle length, and greened cotyledons). A control treatment consisted of maintaining seeds in warm, moist conditions for 12 weeks, which did not break dormancy.</p

Expression analyses and histone H3 methylation pattern changes of regulators of seed maturation/dormancy in Arabidopsis Cvi.

<p>nChIP/qPCR (left column) and expression analyses (right column); averages of three biological replicates are shown +/− SE. Numbers 1–6 correspond to the stages noted on the X-axis for DOG1. ER = endosperm rupture (completion of germination). Note that the Y-axis for the RNA data is in log-scale.</p

Schematic representation of Arabidopsis Cvi seed treatments and physiological status of the seeds.

<p>(<b>A</b>) The inception of primary seed dormancy occurs during seed maturation. Mature dry seeds are dormant (<i>1</i>) and are maintained in this state when imbibed at 22°C, even for 14 d (<i>2</i>). A sufficiently long period of moist chilling (4°C) will break dormancy (<i>3</i>). Light (sun symbol) can induce germination at 22°C once dormancy is broken by moist chilling – seeds commence germination (<i>4</i>), which proceeds to radicle protrusion, signifying the completion of germination (<i>5</i>). The next transition is from germination to seedling growth/development (<i>6</i>). (<b>B</b>) Characterization of seed dormancy of Arabidopsis ecotype Cvi. Moist chilling is required for 14 d to subsequently elicit the full germination potential of seeds. Data are based on mean values of three replicates of 50 seeds +/− SE.</p

Phenotype of <i>fbl17</i> pollen at anther dehiscence.

<p>Pollen from dehiscent anthers of WT and fbl17 ^+/− plants was stained with DAPI and observed under UV illumination. n = total number. Normal pollen all showed two sperm cells. Abnormal pollen showed a single sperm-like cell.</p

<i>fbl17</i> T-DNA insertion mutants.

<p>(A) FBL17 transcript accumulation in plants over-expressing the E2Fa transcription factor and its dimerization partner, DPa. Quantitative RT-PCR on RNA extracted from E2Fa-DPa overexpressing (OE) seedlings show a 15-fold increase in the relative abundance of <i>FBL17</i> transcript compared to control RNA (Col-0). The experiment was three times repeated. Data are means±SE. (B) Diagram of the genomic locus of FBL17. The two T-DNA insertions disrupt the 7^th exon and the 6^th intron in the <i>fbl17-1</i> and <i>fbl17-2</i> allele respectively. Light grey filling indicate non-translated region of the transcript whereas dark grey filling indicates coding sequence. (C) Wild type silique opened to reveal the seed content. (D) Heterozygous <i>fbl17-1^+/−</i> silique displaying a reduced fertility and aborted seeds (marked by white arrows). (E) Homozygous <i>fbl17-1</i> mutant complemented with the <i>FBL17</i> genomic clone show wild type siliques and normal seed development. (C, D, E, bar = 500 µm).</p

Genetic analysis of <i>fbl17</i> mutant plants.

<p>Resistance to sulfadiazine (Sulf^R, sulfadiazine resistant seedlings; Sulf^S, sulfadiazine sensitive seedlings) was used for the <i>AtFbl17-1</i> and <i>AtFbl17-2</i> plants. n = total number. Transmission efficiencies were calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004780#pone.0004780-Howden1" target="_blank">[59]</a>: TE = Sulf^R/Sulf^S×100%.</p

FBL17 interacts with a subset of ASKs.

<p>(A) Yeast-two-hybrid analysis of the interaction between FBL17 and ASKs. Yeast were grown for 3 days at 28°C. LTH- 3AT, low stringency selection, LTA, high stringency selection. Negative controls were done with empty bait vectors (pGBD) or empty prey vectors (pGAD). (B–G) Subcellular interaction of FBL17 with ASK11. Confocal laser-scanning micrographs of the abaxial surface of <i>N. benthamiana</i> leaves. (B, C) Transient expression of ASK11-YFP. The YFP signal is detected both in cytoplasm and nucleus. (D, E) Transient expression of FBL17-YFP. The signal is exclusively nuclear. (F–G) BiFC of FBL17-YN/ASK11-YC. Reconstitution of functional YFP as detected by YFP fluorescence occurs only in the nucleus. (H–K) Subcellular interaction of FBL17 with KRP7. (H, I) Transient expression of KRP7-YFP. A weak YFP signal in the cytoplasm and a strong signal in the nucleus can be detected. (J, K) BiFC of FBL17-YN/KRP7-YC. Reconstitution of functional YFP as detected by YFP fluorescence occurs only in the nucleus. (B, D, F, H, K, J) DIC images of the cells documented. (C, E, G, I, K) laser confocal micrograph of the YFP signal. Scale bars in B to K represent 45 µm.</p

Pollen phenotype of <i>fbl17</i> mutants and <i>FBL17</i> expression during male gametogenesis.

<p><i>fbl17</i> (A) and wild type (B) mature pollen viability test by coloration with Alexander's stain. The purple staining indicates that the grains are viable. Dehiscent pollen of <i>qrt1-1^−/−</i> (C) and <i>qrt1-1^−/−</i>, <i>fbl17-1^+/−</i> (D) stained with DAPI and observed under UV fluorescence. The four pollen grains of the tetrad show two densely stained sperm cell nuclei and one large diffuse vegetative cell nuclei in <i>qrt-1</i> mutants, whereas two pollen grains of the tetrad show only a single germ cell nuclei in <i>qrt1-1,fbl17-1^+/−</i> double mutants. (E, F) transmitted light picture of C and D. (G–L) Expression of the <i>HT10</i> gene in the <i>fbl17</i> mutant pollen. Expression of the HTR10-mRFPprotein under the HTR10 promoter in <i>fbl17-1</i> (H) and wild type (K) pollen, counterstained with DAPI (G, <i>fbl17-1</i>; J, wild type). (I, L) transmitted light pictures of G and J. (A, B) bar = 100 µm; (C–L) bar = 10 µm. (M–U) Promoter-GUS analysis of <i>FBL17</i> expression in pollen. (M, P, S) DAPI staining is applied to reveal the developmental stage of the pollen grain. (N–U) X-Gluc histochemical staining of pFBL17∶GUS (N, Q, F) and non-transformed Col-0 (O, R, U) pollen grains. Bars = 10 µm (V) DNA content measurement of wild type germinative cell nuclei at prophase (n = 9; DNA = 2C), wild type sperm cell nuclei at telophase (n = 16), WT sperm cell nuclei at anthesis (n = 111) in comparison to the unique germ-cell like nuclei of <i>fbl17</i> pollen (n = 77). Error bars = standard error mean.</p