SDG2-Mediated H3K4 Methylation Is Required for Proper <em>Arabidopsis</em> Root Growth and Development

<div><p>Trithorax group (TrxG) proteins are evolutionarily conserved in eukaryotes and play critical roles in transcriptional activation via deposition of histone H3 lysine 4 trimethylation (H3K4me3) in chromatin. Several <i>Arabidopsis</i> TrxG members have been characterized, and among them SET DOMAIN GROUP 2 (SDG2) has been shown to be necessary for global genome-wide H3K4me3 deposition. Although pleiotropic phenotypes have been uncovered in the <i>sdg2</i> mutants, <i>SDG2</i> function in the regulation of stem cell activity has remained largely unclear. Here, we investigate the <i>sdg2</i> mutant root phenotype and demonstrate that <i>SDG2</i> is required for primary root stem cell niche (SCN) maintenance as well as for lateral root SCN establishment. Loss of SDG2 results in drastically reduced H3K4me3 levels in root SCN and differentiated cells and causes the loss of auxin gradient maximum in the root quiescent centre. Elevated DNA damage is detected in the <i>sdg2</i> mutant, suggesting that impaired genome integrity may also have challenged the stem cell activity. Genetic interaction analysis reveals that <i>SDG2</i> and <i>CHROMATIN ASSEMBLY FACTOR-1</i> act synergistically in root SCN and genome integrity maintenance but not in telomere length maintenance. We conclude that SDG2-mediated H3K4me3 plays a distinctive role in the regulation of chromatin structure and genome integrity, which are key features in pluripotency of stem cells and crucial for root growth and development.</p></div

Loss of SDG2 impairs the primary root stem cell niche maintenance. A

<p>and <b>B</b>, Comparison of primary root apical meristem sizes between wild-type <i>Col</i> and the mutant <i>sdg2-3</i>, respectively. DIC images were taken on the roots of 6-day-old seedlings. Arrowheads indicate positions of the transition from meristem to elongation zone. Bar = 100µm. <b>C</b> and <b>D</b>, Comparison of <i>QC25:GUS</i> expression and root cap cell layer organization between <i>Col</i> and <i>sdg2-3</i>, respectively. DIC images were taken on GUS- and Lugol-stained root tips of 6-day-old seedlings. Arrowheads indicate the columella initial cell layer. Bar = 20 µm. <b>E</b> and <b>F</b>, Comparison of cell layer organization of root apical meristem between <i>Col</i> and <i>sdg2-3</i>, respectively. Confocal images were taken on PI-stained roots of 6-day-old seedlings. Bar = 50 µm. The close-up regions are shown by color indication of different cell types: QC cell in blue, columella root cap and columella initial cells in rose, lateral root cap cells in sky-blue, epidermal cells and epidermis/lateral root cap initials in red, cortex cells in green, endodermal cells in yellow, cortex/endodermis initials in purple, stele cells and stele initials in gray. <b>G</b> and <b>H</b>, Comparison of cell layer organizations of root apical meristem between <i>Col</i> and <i>sdg2-3</i>, respectively. Confocal images were taken on PI-stained roots of 14-day-old seedlings. Bar = 50 µm. The close-up regions are shown with colorations as described in <b>E</b> and <b>F</b>.</p

Loss of SDG2 distinctively affects different developmental stages of lateral root formation. A,

<p>Developmental stages of lateral root formation. Images was captured after histochemical GUS staining of roots from 10-day-old <i>Col</i> seedlings expressing <i>DR5:GUS.</i> Developmental stage nomenclature was according to Malamy and Benfey <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056537#pone.0056537-Malamy1" target="_blank">[43]</a>. Bar = 50 µm. <b>B</b>, Relative distribution of developmental stages of lateral root primordia observed in 10-day-old seedlings of the wild-type <i>Col</i> and the mutant <i>sdg2-3</i>. Primordia were counted and examined for developmental stages from at least 20 plants, and the experiments were repeated three times. Mean values of percentage are shown and bars indicate SD.</p

Loss of SDG2 reduces both primary and lateral root growth. A,

<p>Phenotypes of wild-type <i>Col</i> and the mutant <i>sdg2-3</i> seedlings at 26 days after germination. Bar = 1 cm. <b>B</b>, Comparison of primary root length between <i>Col</i> and <i>sdg2-3</i> from 4 to 18 days after germination. <b>C</b>, Comparison of lateral root number between <i>Col</i> and <i>sdg2-3</i> from 8 to 18 days after germination. All data are mean values from two independent experiments with each of at least 20 plants. Bars indicate SD.</p

Loss of SDG2 synergistically enhances growth defects of the CAF1 loss-of-function mutant <i>fas2-4</i>. A

<p>, Representative example of 14-day-old seedling of the wild-type <i>Col</i>, the single mutants <i>sdg2-3</i> and <i>fas2-4,</i> and the double mutant <i>sdg2-3 fas2-4</i>. Bar = 1 cm. <b>B</b>, Comparison of primary root length between <i>fas2-4</i> and <i>sdg2-3 fas2-4</i> on 16-day-old seedlings. Root length is shown as a mean value from two independent experiments with each comprising at least 20 plants. Bar indicates SD. <b>C</b> and <b>D</b>, Comparison of root cap cell organization between <i>fas2-4</i> and <i>sdg2-3 fas2-4</i>, respectively. DIC images were taken on Lugol-stained root tips of 6-day-old seedlings. Arrowhead in <b>C</b> indicates QC position. Bar = 50 µm. <b>E</b> and <b>F</b>, Comparison of cell layer organizations of root apical meristem between <i>fas2-4</i> and <i>sdg2-3 fas2-4</i>, respectively. Confocal images were taken on PI-stained roots of 6-day-old seedlings. The QC cell in <b>E</b> is marked in blue. Bar = 50 µm.</p

Image_1_The Canonical E2Fs Are Required for Germline Development in Arabidopsis.pdf

<p>A number of cell fate determinations, including cell division, cell differentiation, and programmed cell death, intensely occur during plant germline development. How these cell fate determinations are regulated remains largely unclear. The transcription factor E2F is a core cell cycle regulator. Here we show that the Arabidopsis canonical E2Fs, including E2Fa, E2Fb, and E2Fc, play a redundant role in plant germline development. The e2fa e2fb e2fc (e2fabc) triple mutant is sterile, although its vegetative development appears normal. On the one hand, the e2fabc microspores undergo cell death during pollen mitosis. Microspores start to die at the bicellular stage. By the tricellular stage, the majority of the e2fabc microspores are degenerated. On the other hand, a wild type ovule often has one megaspore mother cell (MMC), whereas the majority of e2fabc ovules have two to three MMCs. The subsequent female gametogenesis of e2fabc mutant is aborted and the vacuole is severely impaired in the embryo sac. Analysis of transmission efficiency showed that the canonical E2Fs from both male and female gametophyte are essential for plant gametogenesis. Our study reveals that the canonical E2Fs are required for plant germline development, especially the pollen mitosis and the archesporial cell (AC)-MMC transition.</p

Localized auxin production in tapetum did not rescue the pollen defects in <i>yuc2yuc6</i> mutants.

<p>(A) The green color indicates the expression pattern of the <i>A9</i> promoter. Msp, Microsporocytes; Mp, microspores; Bc, Bicellular pollen; Tc, Tricellular pollen. The <i>A9</i> promoter cloned from Arabidopsis is used to drive <i>YUC2-GFP</i> expression specifically in tapetum cells from stages 6 to 9 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007397#pgen.1007397.ref061" target="_blank">61</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007397#pgen.1007397.ref062" target="_blank">62</a>]. (B) Morphology of adult shoots (Bars = 2cm). (C) Alexander staining (Bars = 100 μm) and DAPI staining (Bars = 10 μm) of Pro<i>A9</i>:<i>YUC2-GFP</i> (<i>yuc2yuc6</i>) anthers and pollens. Note that the sterility phenotype and pollen defects were not rescued in Pro<i>A9</i>:<i>YUC2-GFP</i> (<i>yuc2yuc6</i>) transgenic plants. TS, Tricellular Stage; BS, Bicellular Stage; US; Unicellular Stage. (D) In situ hybridization of GFP in Pro<i>A9</i>:<i>YUC2-GFP</i> (<i>yuc2yuc6</i>) transgenic plants (Bars = 20 μm). (E) Fluorescence images (Bars = 100 μm) of the YUC2-GFP fusion protein in anthers from <i>yuc2yuc6</i> transformed with Pro<i>A9</i>:<i>YUC2-GFP</i>. In Pro<i>A9</i>: <i>YUC2-GFP</i> anther, GFP is significantly expressed in tapetum cell of microsporocyte stage and early microspore stage.</p

Auxin production in diploid microsporocytes is necessary and sufficient for early stages of pollen development

<div><p>Gametophytic development in Arabidopsis depends on nutrients and cell wall materials from sporophytic cells. However, it is not clear whether hormones and signaling molecules from sporophytic tissues are also required for gametophytic development. Herein, we show that auxin produced by the flavin monooxygenases YUC2 and YUC6 in the sporophytic microsporocytes is essential for early stages of pollen development. The first asymmetric mitotic division (PMI) of haploid microspores is the earliest event in male gametophyte development. Microspore development in <i>yuc2yuc6</i> double mutants arrests before PMI and consequently <i>yuc2yuc6</i> fail to produce viable pollens. Our genetic analyses reveal that <i>YUC2</i> and <i>YUC6</i> act as sporophytic genes for pollen formation. We further show that ectopic production of auxin in tapetum, which provides nutrients for pollen development, fails to rescue the sterile phenotypes of <i>yuc2yuc6</i>. In contrast, production of auxin in either microsporocytes or microspores rescued the defects of pollen development in <i>yuc2yuc6</i> double mutants. Our results demonstrate that local auxin biosynthesis in sporophytic microsporocytic cells and microspore controls male gametophyte development during the generation transition from sporophyte to male gametophyte.</p></div

Effects of mutations in <i>YUC2</i> and <i>YUC6</i> on male transmission frequency.

<p>Effects of mutations in <i>YUC2</i> and <i>YUC6</i> on male transmission frequency.</p

The expression patterns of DR5:GFP in the anther of <i>yuc2yuc6</i>.

<p>(A-B) Comparison of the auxin reporter <i>DR5</i>:<i>GFP</i> signal in anthers of wild type (A) and <i>yuc2yuc6</i> (B) at different developmental stages. Reporter signal in anthers observed in wild type from stages 9 to 12 is completely absent in <i>yuc2yuc6</i>. Bars = 100 μm.</p