The ASH1 HOMOLOG 2 (ASHH2) histone H3 methyltransferase is required for ovule and anther development in Arabidopsis

Abstract

Background: SET-domain proteins are histone lysine (K) methyltransferases (HMTase) implicated in defining transcriptionally permissive or repressive chromatin. The Arabidopsis ASH1 HOMOLOG 2 (ASHH2) protein (also called SDG8, EFS and CCR1) has been suggested to methylate H3K4 and/or H3K36 and is similar to Drosophila ASH1, a positive maintainer of gene expression, and yeast Set2, a H3K36 HMTase. Mutation of the ASHH2 gene has pleiotropic developmental effects. Here we focus on the role of ASHH2 in plant reproduction.

Methodology/principal findings: A slightly reduced transmission of the ashh2 allele in reciprocal crosses implied involvement in gametogenesis or gamete function. However, the main requirement of ASHH2 is sporophytic. On the female side, close to 80% of mature ovules lack embryo sac. On the male side, anthers frequently develop without pollen sacs or with specific defects in the tapetum layer, resulting in reduction in the number of functional pollen per anther by up to approximately 90%. In consistence with the phenotypic findings, an ASHH2 promoter-reporter gene was expressed at the site of megaspore mother cell formation as well as tapetum layers and pollen. ashh2 mutations also result in homeotic changes in floral organ identity. Transcriptional profiling identified more than 300 up-regulated and 600 down-regulated genes in ashh2 mutant inflorescences, whereof the latter included genes involved in determination of floral organ identity, embryo sac and anther/pollen development. This was confirmed by real-time PCR. In the chromatin of such genes (AP1, AtDMC1 and MYB99) we observed a reduction of H3K36 trimethylation (me3), but not H3K4me3 or H3K36me2.

Conclusions/significance: The severe distortion of reproductive organ development in ashh2 mutants, argues that ASHH2 is required for the correct expression of genes essential to reproductive development. The reduction in the ashh2 mutant of H3K36me3 on down-regulated genes relevant to the observed defects, implicates ASHH2 in regulation of gene expression via H3K36 trimethylation in chromatin of Arabidopsis inflorescences.

Figure 1. Alleles of ashh2 and the pleiotropic phenotype of ashh2 mutant plants.

(A) Position of the T-DNA insertions of different SALK lines as indicated. The 15 exons of ASHH2 are shown as black boxes and the introns as thin lines. Numbers refer to base pairs (bp) in the Arabidopsis genomic BAC clone T14N5 (acc.no. AC004260) and indicate insertion sites of the T-DNAs, start and stop codons and selected exon-intron borders. The positions of primers used for real-time PCR are show in green above exons 10 and 11. Both the right and the left junction between the ASHH2 gene and the insertions were cloned and sequenced for all alleles except ashh2-5 which is likely to have a complex insertion. Genomic deletions of 33 and 8 bp where found at the insertion sites of ashh2-2 and ashh2-1, respectively. The lower panel shows the ASHH2 protein and the positions of known domains. (B) Relative expression level of ASHH2 in ashh2 mutant plants. Real-time PCR on cDNA from inflorescences of plants homozygous for the indicated alleles as compared to the level in wt inflorescences (set to 1). Standard deviations are shown. (C) Dwarf phenotype of ashh2-1 with several inflorescences (left) compared to wt plant with main inflorescence and a shorter auxiliary shoot (right). (D) Small ashh2-1 cauline leaf (top) and wt cauline leaf (bottom). (E) Small ashh2-1 flower (left) compared to wt flower (right). (F) ashh2-1 flowers with mild (top left) and more severe (bottom left) distorted stamens and carpels compared to wt flowers (right). Some sepals and petals have been removed to display the inner whorl organs. ashh2-1 stamens are often shorter than wt stamens (bottom middle). (G) Siliques of ashh2-1 plant (left) and wt plant (right). (H) ashh2-1 plant 100 days after sowing. (I) Highly branched internode of ashh2-1 plant 100 days after sowing. (J) Composite flower of ashh2-1 plant 100 days after sowing. (K) Scanning electron micrograph (SEM) of ashh2-1 flower with sepals and carpeloid organs with vestigial ovules (ov) and stigmatic papillae (sg). (L) ashh2-1 flower with carpeloid sepals (cs) and stamenoid petals (sp). (M) SEM of stamenoid petal of ashh2-1 flower.

Figure 2. Ovule development and seed set in ashh2 mutant plants, and pASHH2:GUS expression during ovule and seed development.

(A) Total number of seeds/ovules per silique in selfed wt (n _siliques = 12), homozygous (n _siliques = 125) and heterozygous ashh2-1 plants (n _siliques = 26). (B) Percentage of developing green seeds and undeveloped ovules per silique in selfed wt (n _siliques = 12), homozygous (n _siliques = 125) and heterozygous ashh2 plants (n _siliques = 26) prior to seed desiccation. (C) In situ hybridization on longitudinal sections of seeds at the heart stage (left) and the walking stick stage (right) of embryo development with ASHH2 antisense probe (AS) and control sense probe (S). Expression was seen in the whole heart stage embryo (em), the chalazal endosperm (cze) as well as the endosperm nodules (en) aligning the embryo sac around the vacuole (v). At the walking stick stage expression was seen both in the cotyledons (cot), hypocotyl (hyp) and root tip (r). (D) Full-length ashh2-1 mutant silique compared to a segment of a wt siliques, with enlarged sections to the right. Arrows point to undeveloped ovules. (E) pASHH2:GUS expression in the nucellus of the developing ovule. The GUS signal is located in the L2 layer where the megaspore (m) forms. Nucellar tissue (n), chalazal tissue (cz), inner integuments (ii), outer integuments (oi). (F) pASHH2:GUS expression in the ovule with mature embryo sac. Strong expression is found in maternal chalazal proliferating tissue (cz). Weaker expression was observed in the nucellus and inner (ii) and outer integuments (oi). (G) pASHH2:GUS expression in maternal tissue strongly in the chalazal tissues (cz) and weaker in the inner integuments (ii) and even weaken in the outer integument (oi) after fertilization. The chalazal region (cz) is indicated by an arrow and pollen tubes (pt) that have grown along the funiculus (f) with thin lines. The septum (s) with its transmitting tract (tt) is indicated.

Figure 3. Ovule and embryo sac development in wt and ashh2-1.

Differential interference contrast (DIC) micrographs of (A) wt and (B–L) ashh2-1 ovules at one day post anthesis (A–C), anthesis (D–F), day before anthesis (G–I) and at the time point of the first syncytial nuclear division (J–L). Scale bar, 25 µm. (A) Wt mature ovule harboring embryo sac with (arrows) egg cell (ec), central cell (cc) and synergid cells (syn, only one synergid cell is in the focal plane). The embryo sac is surrounded by maternal chalazal (cz) tissue and endothelium layer (et), and further surrounded by two more layers of inner integuments (ii) and two layers of outer integuments (oi). Arrowheads mark the border between the embryo sac and the adaxial (adx) endothelium and the abaxial (abx) chalazal tissue. (B) ashh2-1 ovule without embryo sac between endothelium and the adaxial chalazal tissue (arrowheads). Ovule morphology appears fairly normal with two outer integument layers (oi) and three inner integuments (ii and et). The chalazal tissue in the center of the ovule appears to have proliferated more than in wt. (C) ashh2-1 ovule from same silique as (B) with a single mono-nucleate cell (boxed). Inset in the upper right corner shows magnification of boxed area (n, nucleus). The outer integuments engage both inner integuments and nucellus. (D) ashh2-1 syncytial embryo sac with eight nuclei. Inset in the upper right corner shows magnification of boxed area (n, nucleus). Two nuclei are not in the focal plane. (E) Proliferated ashh2-1 syncytial embryo sac. Inset in the upper right corner is a merge of two focal planes and shows magnification of the boxed area (n, nucleus). The basally located nuclei appear to degenerate. Note that the micropylar end of the embryo sac is not covered by integuments in both (E) and (F). (F) ashh2-1 embryo sac. Inset in the upper right corner shows magnification of the boxed area show two nuclear structures reminiscent of egg and central cell nuclei (n, nucleus). (G–H) ashh2-1 ovules with embryo sac-like structure (arrow) budding off the micropylar end of the ovule. The integuments do not encase the structure. (I) ashh2-1 ovule arrested or delayed in development. The inner integuments have developed longer than the outer integuments. (J–L) ashh2-1 ovules with two nucleate syncytial embryo sacs. No vacuole could be observed between the nuclei (n, nucleus).

Figure 4. Pollen development in ashh2 and pASHH2:GUS expression in anther development.

(A) Average number of pollen per anther (n_wt = 4, n_ashh2>8) in wt and ashh2 mutant lines. Standard deviations are shown. (B) Light- and fluorescent micrographs of ashh2-1 anthers and germinating pollen tubes. (i) Four long stamina from ashh2-1 from a flower just before abscission. All anthers are delayed in dehiscence. Note different numbers of pollen (p) in each locule and absence of normal locule development (arrows). (ii) Mature ashh2-1 anther with released pollen in the trinucleate stage (boxed area). Inset is a magnification of the boxed area, (s) sperm cell, (v) vegetative cell. (iii) Pollen tube germination on the papillae in ashh2-1. (p) pollen, (pt) pollen tube. (C) Stage 7 flower of pASHH2:GUS plant. No GUS expression was seen in the developing floral organs but only in the pedicel. s – sepal; g – gynoecium; pp – petal primordium; sp – stamen primordium. (D) Stage 9 to 11 flowers showing increasing GUS expression post meiosis. The boxed insert shows that no GUS expression was detected in tetrads. s – sepal; g – gynoecium; a – anther. (E) Stage 12 flower with mature anthers with GUS expression in anthers (a), as well as the gynoecium (g) and mid veins of petals (p) and sepals (s). (F) Anther of stage 12 flower showing GUS expression both in the tapetal cell layer (t) and the mature microspores (ms), but not in the anther filaments (f). (G) Detail of GUS expression in floral stage 12 anther demonstrate specific expression in tapetum (t) and microspores (ms), but not in the surrounding endothecium (en) and epidermis (e) cell layers. (H) Scanning electron micrographs of wt pollen (i) with a sexine layer (ii) with regular ridges (muri – m) and spaces (lumina – l). (I) Scanning electron micrographs of ashh2-1 pollen grains (i, ii) and exine layers with filled muris (iii), and in severe cases visible nexine layer (n) and bacula (b) due to absent tectum layer (iv).

Figure 5. TEM analysis of tapetal and pollen development in ashh2 and wt.

(A) Early Ring Stage with free microspores (M) in wt. Note the endothecium (E), tapetum (T) and intact middle cell layer (mc). (B) Pollen Mitosis I to Pollen Mitosis II in wt, degeneration of the middle cell layer has occurred. Note exine (ex) with visible defined bacula. (C) Immediately prior to dehiscence in the wt. Tapetal degeneration and deposition of pollen coat has occurred and endothecium (E) secondary thickening is visible. (D) Early Ring Stage in the ashh2-1 mutant displaying abnormal tapetal (T) enlargement. (E) Pollen Mitosis I to Pollen Mitosis II of ashh2-1 disclosing abnormal accumulations of lipid-like material in membrane bound vesicles (arrows) within the tapetal cells (T). Note that normal tapetal degeneration fails to occur and that the tapetal membrane is clearly visible (arrowhead). (F) Prior to dehiscence in the ashh2-1 mutant, normal endothecium secondary thickening (st) and thinning of the stomium (S) is seen. However the tapetal cells (T) are still present and release of the globular material previously visible (Figure 6E) has not occurred, suggesting that tapetal development is impaired and delayed. (G and J) Pollen Mitosis I to Pollen Mitosis II in the ashh2-2 mutant, note secondary thickening (st) of the endothecium and that degeneration of the middle cell layer has occurred. Exine formation (ex) with well-defined bacula is visible, however, the tapetum (T) appears enlarged with an abnormal accumulation of wall materials. (H and K) As pollen mitosis progresses in ashh2-2 abnormal deposition of pollen wall material is evident (arrow), resulting in agglutination of the immature pollen grains. In some cases a deficiency of the exine sculpturing is observed (arrowheads). (I and L) Prior to dehiscence in ashh2-2 normal breakdown of the stomium occurs, however abnormal pollen wall development is observed (arrow), resulting in some cases in extreme malformation of the pollen wall (L). Bars in A, H: 10 µm; C, D, E, G, L: 5 µm; B, J, K: 2 µm; F, I: 20 µm.

Figure 6. Changes in gene expression and histone tail methylation in the ashh2-1 mutant.

(A) Functional GO annotation of genes differentially expressed in ashh2-1 inflorescences relative to wt. The inserts depict the developmental stage of the main inflorescences used in the microarray experiment. (B) Number of co-down-regulated genes in the ashh2-1 mutant (>1.6-fold change), ms1 mutant (>2-fold change) and spl/ems-1 mutant (>1.6-fold change). (C) Confirmation of down-regulation of selected genes using real-time reverse transcriptase PCR. Wt levels were set to 1. (D) Gene structures of AtDMC1, AP1 and MYB99. Boxes indicate exons, thin lines introns. The positions of the fragments tested in the ChIP analyses are shown as red lines. (E) Representative results of ChIP analyses on the selected down-regulated genes shown in (D) using antibodies against H3K4me3, H3K36me2 and H3K4me3. Ta3 was used as a control. IN – input; –ab – without antibodies.