Plant Physiology Preview. Published on April 28, 2017, as DOI:10.1104/pp.17.00306
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Running head:
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Crucial function of SDG2 in male gametogenesis
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Corresponding author:
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Wen-Hui Shen
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Université de Strasbourg
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CNRS, IBMP UPR2357
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F-67000 Strasbourg
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France
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Phone +33 3 67 15 53 26
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Fax +33 3 67 15 53 00
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email [email protected]
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Copyright 2017 by the American Society of Plant Biologists
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SDG2-mediated H3K4me3 is crucial for chromatin condensation and mitotic division during
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male gametogenesis in Arabidopsis [1]
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Violaine Pinon 2, Xiaozhen Yao2, Aiwu Dong , Wen-Hui Shen *
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Université de Strasbourg, CNRS UPR2357, F-67000 Strasbourg, France (V.P., W.-H.S.); State
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Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and
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Development, International Associated Laboratory of CNRS-Fudan-HUNAU on Plant
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Epigenome Research, Department of Biochemistry, Institute of Plant Biology, School of Life
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Sciences, Fudan University, Shanghai 20043, PR China (X.Y., A.D. W.-H.S.); College of Life and
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Environment Sciences, Shanghai Normal University, Shanghai 200234, PR China (X.Y.)
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Footnotes:
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0013-02), the National Basic Research Program of China (973 Program, 2012CB910500 and
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2011CB944600), the National Natural Science Foundation of China (grant no. 31571319,
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31371304 and 31300263), the Science and Technology Commission of Shanghai Municipality
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(grant no. 13JC1401000), and the French Centre National de la Recherche Scientifique
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(CNRS, LIA PER)
This work was supported by the French Agence Nationale de la Recherche (ANR-12-BSV2-
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These authors contributed equally to this work.
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*Corresponding author; e-mail [email protected]
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Authors' contributions: W.-H.S. conceived the original research plans and supervised the
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experiments; V.P. and X.Y. performed the experiments and analyzed the data with equal
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contribution; A.D. provided assistance to X.Y. and supervised some of the experiments; V.P.,
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X.Y. and W.-H.S. wrote the article with contributions of all the authors.
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One-sentence summary: Active H3K4me3 deposition is essential for gametophyte chromatin
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landscape, playing critical roles in gamete mitotic cell cycle progression and pollen
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vegetative cell function.
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ABSTRACT
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Epigenetic reprogramming occurring during reproduction is crucial for both animal and plant
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development. Histone H3 lysine 4 trimethylation (H3K4me3) is an evolutionarily conserved
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epigenetic mark of transcriptional active euchromatin. While much has been learned in
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somatic cells, H3K4me3 deposition and function in gametophyte is poorly studied. Here we
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demonstrate that SDG2-mediated H3K4me3 deposition participates in epigenetic
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reprogramming during Arabidopsis male gametogenesis. We show that loss of SDG2 barely
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affects meiosis and cell fate establishment of haploid cells. However, we found that SDG2 is
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critical for postmeiotic microspore development. Mitotic cell division progression is partly
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impaired in the loss-of-function sdg2-1 mutant, particularly at the second mitosis setting up
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the two sperm cells. We demonstrate that SDG2 is involved in promoting chromatin
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decondensation in the pollen vegetative nucleus, likely through its role in H3K4me3
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deposition, which prevents ectopic heterochromatic H3K9me2 speckle formation. Moreover,
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we found that derepression of the LTR retrotransposon ATLANTYS1 is compromised in the
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vegetative cell of the sdg2-1 mutant pollen. Consistent with chromatin condensation and
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compromised transcription activity, pollen germination and pollen tube elongation,
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representing the key function of the vegetative cell in transporting sperm cells during
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fertilization, are inhibited in the sdg2-1 mutant. Taken together, we conclude that SDG2-
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mediated H3K4me3 is an essential epigenetic mark of the gametophyte chromatin
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landscape, playing critical roles in gamete mitotic cell cycle progression and pollen
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vegetative cell function during male gametogenesis and beyond.
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INTRODUCTION
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In animals and plants, epigenetic reprogramming during reproduction is believed to play
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crucial roles in maintaining genome integrity, in potentiating genetic and epigenetic
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variation, in resetting gene-expression program, and in contributing to the embryo
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totipotency at fertilization (Sasaki and Matsui 2008; Feng et al. 2010; Kawashima and Berger
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2014; Lesch and Page 2014). In animals, a diploid germline arises early during embryogenesis
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and remains as a distinct niche of stem cells (primordial germ cells) throughout life (Sasaki
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and Matsui 2008). In flowering plants, however, the germline is differentiated from somatic
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cells within specialized organs of the flower, which is formed late in development, after a
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long postembryonic phase of plant growth (Ma 2005). Furthermore, while in animals
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gametes are direct products of meiosis, in plants multicellular haploid gametophytes are
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formed through rounds of postmeiotic cell divisions. Thus, in many flowering plants
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including the model plant Arabidopsis thaliana, the male gametophyte is characterized by a
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3-celled pollen and the female gametophyte by a 7-celled embryo sac (Berger and Twell
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2011; Schmid et al. 2015). Upon double fertilization, one of the two haploid sperm cells from
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the pollen fuses with the egg cell of the embryo sac to form the diploid zygote, which
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develops into embryo. The other sperm cell fuses with the central cell of the embryo sac to
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produce the triploid endosperm, a tissue providing nourishment to the developing embryo
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(Hamamura et al. 2012). These differences of reproductive strategies used by plants as
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compared to animals are driving intense interest on epigenetic reprogramming mechanisms
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associated with complex cell fate establishment and maintenance during plant reproduction.
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Cell-type specificities lay in distinctive transcriptomes that are controlled by the
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activity of transcription factors, and by the chromatin structure, which can impede the
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accessibility and activity of the transcriptional machinery. In mammals, an erasure of almost
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all DNA methylation states is observed before fertilization (Feng et al. 2010). In plants,
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however, such extreme reprogramming event does not seem to occur at fertilization
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(Ingouff et al. 2017; Saze et al. 2003; Jullien et al. 2012; Calarco et al. 2012). Moreover, over
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85% of mammalian histones are replaced by sperm-specific protamine proteins enabling
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extensive compaction of DNA in male gametes, and upon fertilization the paternal
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chromatin is deprived of the protamines and loaded with variant histone H3.3 provided by
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the female gametes (Ooi and Henikoff 2007; Saitou and Kurimoto 2014). However, plants do
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not contain protamines, and in Arabidopsis germ cells the canonical histone H3.1 is replaced
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by the H3.3 variant, HISTONE THREE RELATED10 (HTR10), which is subsequently removed
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from the zygote nucleus upon fertilization (Ingouff et al. 2007; Ingouff et al. 2010). Within
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pollen, chromatin is less condensed in the vegetative cell nucleus than in the two sperm cell
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nuclei. Interestingly, during microsporogenesis, DNA demethylation occurs in the pollen
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vegetative cell nucleus and activation of transposable elements (TEs) in the vegetative cell
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has been proposed to generate small RNAs to reinforce silencing of complementary TEs in
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sperm cells (Ibarra et al. 2012; Slotkin et al. 2009; Calarco et al. 2012). Moreover, the
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heterochromatin mark H3K9me2 specifically accumulates in the two sperm cell nuclei while
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it is greatly absent from the vegetative cell nucleus (Schoft et al. 2009), and the other
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histone methylations including H3K4me2/me3, H3K36me2/me3 and H3K27me3 are
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detected abundantly in the vegetative cell nucleus (Cartagena et al. 2008; Sano and Tanaka
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2010; Houben et al. 2011; Pandey et al. 2013).
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Methylation of histone lysine residues depends on the activity of Histone
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Methyltransferases (HMTases): most of them contain an evolutionarily conserved catalytic
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SET-domain (Huang et al. 2011; Thorstensen et al. 2011; Herz et al. 2013). So far, various
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HMTases have been characterized in animals and plants, primarily in somatic cells but rarely
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in germ cells.
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ATX1/SDG27, ATX2/SDG30, ATXR7/SDG25 and ATXR3/SDG2, have been reported to catalyze
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H3K4 methylation. Disruption of ATX1 causes pleiotropic phenotypes including homeotic
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transformation, root and leaf defects, and early flowering, whereas loss of ATX2 alone does
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not have an obvious phenotype but can enhance atx1 mutant defects (Alvarez-Venegas et al.
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2003; Pien et al. 2008; Saleh et al. 2008). The loss-of-function sdg25/atxr7 mutant has an
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early flowering phenotype associated with suppression of FLOWERING LOCUS C (FLC)
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expression (Berr et al. 2009; Tamada et al. 2009). Remarkably, ATXR3/SDG2 is broadly
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expressed and its disruption leads to severe and pleiotropic phenotypes including dwarfism,
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short-root, impaired fertility and altered circadian clock, as well as the misregulation of a
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large number of genes (Berr et al. 2010; Guo et al. 2010; Malapeira et al. 2012; Yun et al.
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2012; Yao et al. 2013).
In Arabidopsis, several SET DOMAIN GROUP (SDG) proteins, including
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In this study, we investigate the function of SDG2 in chromatin reprogramming
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during male gametophyte development. While loss of SDG2 does not show detectable effect
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on meiosis and cell fate acquisition, the loss-of-function sdg2-1 mutant displays a pause or
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delay of developmental progression at bicellular pollen stage. Chromatin condensation was
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found elevated in the pollen vegetative cell nucleus, which is in association with a reduction
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of H3K4me3 and an ectopic speckle formation of the heterochromatin mark H3K9me2
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detected in the sdg2-1 pollen grains. Remarkably, in the sdg2-1 mutant, the activation of
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retrotransposon ATLANTYS1 in the pollen vegetative cell is impaired, and pollen germination
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as well as pollen tube elongation is inhibited. Our study thus establishes a crucial role of
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SDG2-mediated H3K4me3 in chromatin landscape regulation and postmeiotic microspore
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development and function.
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RESULTS
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Loss of SDG2 barely affects male meiosis
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Arabidopsis SDG2 has been shown to be essential in sporogenous tissues during specification
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of floral organs, as well as in gametophytic function (Berr et al. 2010; Guo et al. 2010). In
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these previous studies, several independent sdg2 mutant alleles had been characterized to
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display similar loss-of-function mutant phenotype, and the mutant phenotype could be fully
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rescued to wild-type phenotype in complementation test. It was reported that
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independently from flower developmental defects, the probability of the sdg2-1, sdg2-2 or
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sdg2-3 allele transmission from the selfing population in heterozygous (+/-) mutant plants is
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reduced roughly to 55-75%. Furthermore, analyses of reciprocal crosses between the control
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wild-type Columbia-0 (Col-0) and the heterozygous mutant sdg2-1(+/-) plants or between
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Col-0 and sdg2-3(+/-) plants showed that both male and female transmission are affected
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(Berr et al. 2010). The decreased transmission of mutant alleles does not result from an
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increase in embryo lethality (Berr et al. 2010). This phenotype might rather result from
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defects in meiosis progression and/or gametophyte development. To investigate those
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possibilities, we first monitored the different stages of meiosis in sdg2-1 mutant using 4, 6-
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diamidino-2-phenylindole (DAPI) staining of microspore mother cells. Meiosis consists of two
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rounds of cell divisions after a single round of DNA replication, producing haploid gametes.
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The meiosis events have been clearly described previously (Ross et al. 1996; Ma 2005); the
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different meiotic stages observed in the control Col-0 plants are shown in Fig. 1A to 1J.
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During meiosis I in sdg2-1, the condensed homologous chromosomes form an array on the
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nuclear plate following synapsis and pairing in prophase I (Fig. 1K to 1M), similarly as in Col-0
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(Fig. 1A to 1C). The homologous chromosomes then migrate to opposite poles in sdg2-1 (Fig.
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1N and 1O) similarly as in Col-0 (Fig. 1D and 1E), and after meiosis II, individual sister
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chromatids are separated to form tetrad of haploid nuclei in sdg2-1 (Fig. 1P and 1T) similarly
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as in Col-0 (Fig.1F to 1J). Based on the observation of more than 50 meiocytes at each
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meiotic stage, we found that the meiosis events in the sdg2-1 mutant show no significant
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differences compared to Col-0. To conclude we could not detect any abnormal chromosome
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configuration during meiosis in sdg2-1, suggesting that SDG2 activity rather regulates
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postmeiotic gametogenesis in Arabidopsis.
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SDG2 is not essential for establishment of pollen vegetative and sperm cell identity
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To further characterize SDG2 function during postmeiotic gametogenesis, we investigated
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the establishment of gametophytic cell fate in sdg2-1 mutant. We analyzed postmeiotic
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events using specific vegetative and sperm cell nuclei reporters, respectively AC26-mRFP and
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HTR10-mRFP, combined with nuclear DAPI staining. The living-cell reporter HTR10-mRFP
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represents a fusion between the H3.3 variant HTR10 protein and the fluorescent protein
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mRFP under the control of the native HTR10 promoter (Ingouff et al. 2007); and AC26-mRFP
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is associated with the expression of HISTONE H2B-mRFP fusion protein under the control of
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ACTIN11 promoter (Rotman et al. 2005). Analyses under the confocal microscope revealed
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that HTR10-mRFP specifically marks the generative cell nucleus of bicellular pollen (Fig. 2A)
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and the two sperm cell nuclei of tricellular pollen (Fig. 2B) in a way similar between Col-0
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and sdg2-1. Also the vegetative cell marker AC26-mRFP was observed specifically marking
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the vegetative cell within the mature pollen in a way similar between Col-0 and sdg2-1 (Fig.
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2C). Our data thus indicate that the establishment of sperm and vegetative cell fate can
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occur normally in sdg2-1 pollen. Furthermore our observation also indicates that specific
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incorporation of the HTR10 variant before fertilization in the chromatin of generative and
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sperm cell nuclei (Ingouff et al. 2007) is unaffected in sdg2-1, as compared to wild-type.
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SDG2 is involved in the timing of pollen development
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To further understand sdg2-1 pollen defects that lead to a reduced transmission of the
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mutant allele, we analyzed the developmental timing of pollen maturation. Anther
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formation in Arabidopsis is divided into 14 developmental stages (Sanders et al. 1999). The
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meiosis that will generate the microspores occurs around stage 6 of anther development.
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While the first mitotic division takes place at stages 11, the second division that will produce
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tricellular pollen is observed at anther developmental stages 12. We first examined bicellular
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stage pollen (anther stage 11) using HTR10-mRFP marker to count the number of
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generative/sperm nuclei per pollen. In both Col-0 and sdg2-1, more than 94% of pollen
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grains contained one generative nucleus per pollen as expected for normal development
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(Fig. 3A). Later at development, in tricellular stage pollen (anther stages 12-13), 82% of the
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Col-0 pollen grains had two sperm nuclei per pollen marked with HTR10-mRFP, while only
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65% of the sdg2-1 pollen grains showed two sperm nuclei per pollen, and 24% of the sdg2-1
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pollen grains showed one sperm nucleus per pollen (versus 11% in Col-0) (Fig. 3B). To verify
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that sdg2-1 pollen from anther stages 12-13 with one HTR10-mRFP positive nucleus is
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indeed at bicellular stage, we also used DAPI staining in examination. We found that sdg2-1
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pollen with one nucleus marked with HTR10-mRFP is always a bicellular pollen and never a
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tricellular pollen (Fig. 3C). Therefore we conclude that the transition from bicellular to
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tricellular stage of pollen development is slowing down or paused in sdg2-1.
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SDG2 is involved in maintenance of decondensed chromatin state of pollen vegetative cell
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In both male and female gametophytes, specific cell types are characterized by specific
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chromatin organization. In wild-type Arabidopsis pollen, the vegetative cell nucleus contains
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a relatively decondensed chromatin whereas the sperm nuclei contain highly condensed
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chromatin (Schoft et al. 2009). In sdg2-1 mutant, global chromatin organization might be
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impaired, and would then affect pollen development. To verify this hypothesis, we analyzed
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DNA condensation in pollen vegetative and sperm cell nuclei using DAPI staining. Confocal
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analyses showed that at early stage of gametophyte development, DNA in haploid
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microspore is more condensed in sdg2-1 than in Col-0 (Fig. 4A, upper panels). At the
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following bicellular and tricellular stages of pollen development, we found that DNA
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condensation remained obviously higher in the vegetative cell nuclei of sdg2-1 as compared
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to those of Col-0 (Fig. 4A, middle and lower panels; and Fig. 4B and 4C). In contrast,
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significant differences in DNA condensation were undetectable in generative or sperm cell
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nuclei between sdg2-1 and Col-0 (Fig. 4). Together, our data indicate that SDG2 is required
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for proper chromatin decondensation in the microspore and later on in the vegetative cell
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nucleus at bicellular and tricellular stages of pollen development.
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SDG2-mediated H3K4me3 prevents ectopic heterochromatic H3K9me2 speckle formation
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in pollen nuclei
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To confirm that SDG2 is indeed involved in regulating H3K4me3 in pollen, we utilized anti-
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H3H4me3 antibodies to immunolocalize the histone mark in wild-type versus mutant pollen.
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We found that H3K4me3 is present in both vegetative and sperm nuclei of Col-0 pollen (Fig.
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5A, upper panels) and its signal intensity are greatly reduced in both vegetative and sperm
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nuclei of the sdg2-1 mutant pollen (Fig. 5A, lower panels) compared to Col-0 pollen (Fig. 5B).
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Western blot analysis revealed that H3K4me3 level is globally reduced in the sdg2-1
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inflorescence (Fig. 5C). These data are consistent with the previous reports showing that
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SDG2 encodes a major H3K4-methyltransferase in Arabidopsis (Berr et al. 2010; Guo et al.
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2010). In contrast to the euchromatin marker H3K4me3, H3K9me2 represents the
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heterochromatin marker in Arabidopsis. Our western blot analysis failed to detect any clear
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changed level of H3K9me2 in the sdg2-1 inflorescence (Fig. 5C). Our immunolocalization
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analysis showed that H3K9me2 is detected in speckles in the sperm cell nuclei but barely in
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the vegetative nucleus (<9%, n=56) of Col-0 pollen (Fig. 5D and 5E; Supplemental Fig. S1),
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which is in agreement with a previous study (Schoft et al. 2009). Interestingly, the sdg2-1
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mutant pollen showed clearly detectable H3K9me2 speckles in the vegetative nucleus
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(>30%, n=52) as well as an increase of speckles in the sperm nuclei (Fig. 5D and 5E;
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Supplemental Fig. S1). Examination of pollen at bicellular stage also revealed an increase of
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H3K9me2 speckles in both vegetative and generative nuclei in sdg2-1 as compared to Col-0
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(Fig. 5F; Supplemental Fig. S1). From those data, we conclude that SDG2-mediated H3K4me3
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prevents ectopic heterochromatic H3K9me2 speckle formation, and that the reduced level
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of H3K4me3 together with H3K9me2 redistribution likely has caused high chromatin
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compaction observed in the vegetative nucleus of the sdg2-1 mutant.
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SDG2 is required for active expression of retrotransposon ATLANTYS1 in pollen
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Previous studies have revealed that global DNA demethylation specifically occurs in the
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pollen vegetative cell nucleus, and active transcription and production of mobile siRNAs
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from TEs in the vegetative cell are proposed to act on complementary TE silencing
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associated with compact chromatin in the sperm cell nuclei (Ibarra et al. 2012; Slotkin et al.
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2009; Calarco et al. 2012). The presence of a more condensed DNA together with the
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decreased H3K4me3 in sdg2-1 might affect TEs derepression in the pollen vegetative cell. To
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directly analyze the functional effect of chromatin condensation defect in sdg2-1 pollen, we
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crossed sdg2-1/+ with a GUS enhancer trap line ET5729, which contains an insertion of β-
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glucuronidase (GUS) coding sequence in an endogenous retrotransposon ATLANTYS1
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(Sundaresan et al. 1995; Slotkin et al. 2009). To eliminate possible mixed genetic background
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effect, we also crossed Col-0 with ET5729 (Landsberg erecta, Ler background) and used as
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control. GUS staining analysis revealed that wild-type Col-0 pollen is absent of staining (Fig.
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6A) and pollen from F2 plants of Col-0 x ET5729 shows clear blue GUS staining (Fig. 6B).
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Interestingly, pollen from F2 plants of sdg2-1 (Col-0 background) x ET5729 showed barely
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detectable GUS blue staining (Fig. 6C). Consistently, at later generation, with the GUS
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reporter allele at homozygous state, strong blue staining was detected in ET5729 (Col-0)
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control pollen (Fig. 6D) but not in ET5729 (sdg2-1) mutant pollen (Fig. 6E). Thus, our data
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indicate that ATLANTYS1 activation is impaired in sdg2-1. Analysis of the previously
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published microarray data (Berr et al. 2010) revealed that 20 TE genes are down-regulated
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by more than 2-fold in sdg2-1 compared to Col-0 flower buds (Supplemental Table S1).
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Transcripts of the majority of these TE genes are detectable in the male meiocyte (Yang et al.
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2011) and/or in pollen (Loraine et al. 2013) by RNA sequencing (RNA-seq) analysis. More
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notably, among these TE genes At5g28626 corresponds to a full-length SADHU element,
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which is disrupted by an ATLANTYS2-like retrotransposon LTR sequence and controlled in
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silencing by DNA methylation in some Arabidopsis accessions (Rangwala et al. 2006).
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Collectively, the data indicate that transcription activation of various TE genes relies on
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SDG2, which is in agreement with the reduced H3K4me3 and high chromatin compaction
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observed in the vegetative cell of sdg2-1 pollen.
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SDG2 plays a critical role in pollen germination and pollen tube elongation
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A major function of pollen vegetative cell activity is to assure pollen germination and
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subsequent pollen tube elongation to deliver sperm cells for fertilization. Lastly, we
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addressed the question whether or not SDG2 plays a role in these processes. Thus we
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carried out in vitro pollen germination assays. After 8 hours incubation on appropriate
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culture medium, nearly 77% of the Col-0 pollen grains produced pollen tubes (n=300), while
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the germination ratio of the sdg2-1 mutant pollen grains arrived only to about 10% (n=300)
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(Fig. 6F,G). Moreover, the length of the mutant pollen tube was drastically inhibited, as
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compared to the good length of wild-type pollen tube (Fig. 6F). These data clearly
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demonstrate that loss of SDG2 impairs pollen germination and pollen tube elongation, which
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is in line with high chromatin compaction and compromised transcription activity observed
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in the sdg2-1 pollen vegetative cell.
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DISCUSSION
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In this study we show that SDG2-mediated H3K4me3 participates to the epigenetic
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reprogramming observed during Arabidopsis male gametogenesis. Although male meiosis
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and cell fate acquisition can occur normally in the sdg2-1 mutant, SDG2-mediated H3K4me3
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is crucial in promoting postmeiotic microspore chromatin decondensation, mitotic cell
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division, and activation of retrotransposon ATLANTYS1. Consistent with a compromised
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vegetative cell function, pollen germination and pollen tube elongation are found inhibited
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in the sdg2-1 mutant.
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In wild-type Arabidopsis, as compared to surrounding somatic cells, both
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megaspore and microspore mother cells display more decondensed chromatin with higher
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levels of H3K4me2 and H3K4me3 (She et al. 2013; She and Baroux 2015). The MALE
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MEIOCYTE DEATH 1 (MMD1, also known as DUET) gene encodes a PHD-finger protein
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essential for male meiosis (Reddy et al. 2003; Yang et al. 2003). Loss of MMD1/DUET
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function causes multiple defects including cytoplasmic collapse and cell death of meiocytes
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with possible DNA fragmentation, delay in progression and arrest at metaphase I, and
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formation of aberrant meiotic products such as dyads and triads (Reddy et al. 2003; Yang et
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al. 2003; Andreuzza et al. 2015). More recent studies demonstrate that the MMD1/DUET
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PHD-finger exhibits binding activities to modified histone H3 peptides including H3K4me2
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and H3K4me3 and that its mutation compromises MMD1/DUET biological function
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(Andreuzza et al. 2015; Wang et al. 2016). Based on these previous studies, one can predict
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that H3K4me2/me3 might play crucial roles in Arabidopsis male meiosis. In contrast,
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however, depletion of the Arabidopsis major H3K4me2/me3-methyltransferase SDG2 in the
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sdg2-1 mutant (Berr et al. 2010; Guo et al. 2010) showed normal male meiosis without any
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detectable chromosomal abnormality throughout different meiotic phases (Fig. 1). The
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female meiosis also occurs normally in the sdg2-1 mutant, albeit reduced H3K4me3 in
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megaspore mother cells (She et al. 2013). It seems that Arabidopsis meiosis is rather tolerant
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to H3K4me3 reduction. Nevertheless, cautions are necessary because Arabidopsis contains
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multiple H3K4-methyltransferases that may have overlap/redundant function in meiosis.
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Transcripts of SDG2 as well as those of ATX1, ATX2 and SDG4 have been detected in male
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meiocytes (Yang et al. 2011). Moreover, SDG4 has been reported to contribute to pollen
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function, likely through methylation of H3K4 and H3K36 (Cartagena et al. 2008). Future
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studies will be required to verify possible overlap/redundant functions of these different
338
HMTase genes to better understand the role of H3K4me2/me3 deposition in Arabidopsis
339
meiosis.
340
After meiosis II, sister chromatids are separated to form tetrad of haploid cells in
341
sdg2-1 as in Col-0 (Fig. 1P and 1T). Nevertheless, tetrad analysis had revealed significant loss
342
of nuclei in sdg2-1 (Berr et al. 2010), indicating that chromatin in the mutant might be
343
unstable and DNA degradation might have occurred in some mutant microspores. During
344
gametogenesis, the first haploid cell division leads to establishment of the vegetative cell
345
and the generative cell at bicellular pollen stage, and the second mitotic cell division occurs
346
specifically from the generative cell resulting in tricellular pollen containing the vegetative
347
cell and two sperm cells. Our analysis of living pollen grains shows that cell fate acquisition
348
can occur normally in the sdg2-1 mutant (Fig. 2). However, SDG2-depletion prevents the
349
second pollen mitosis, generating a fraction of pollen grains comprising a vegetative cell and
350
a single sperm cell (Fig. 3). One major regulator of sperm cell identity and cell cycle
351
progression is the transcription factor DUO POLLEN1 (DUO1) (Rotman et al. 2005; Borg et al.
352
2011; Borg et al. 2014). In the loss-of-function mutant duo1, the generative cell fails to
353
divide to generate two sperm cells, and the mutant pollen cannot fertilize the female egg
354
cell or central cell. Specific expression of the histone variant gene HTR10 in sperm cells is
355
under the direct control of DUO1 (Borg et al. 2014). Our study shows that HTR10-mRFP
356
expression is unaffected in the sdg2-1 pollen (Fig. 2 and Fig. 3), suggesting that SDG2 acts
357
independently from the DUO1-pathway. The more condensed chromatin observed in the
358
vegetative cell (Fig. 4) might have affected its genome function and indirectly impairs
359
generative cell division in the sdg2-1 pollen. The histone chaperone CHROMATIN ASSEMBLY
360
FACTOR1 (CAF1), which is involved in de novo assembly of nucleosomes in a replication-
361
dependent manner, is also required for mitotic cell division but not for cell fate maintenance
362
during pollen development (Chen et al. 2008). Together those data suggest the existence of
363
a strong link between the chromatin landscape and cell cycle progression for proper pollen
364
development.
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365
Highly condensed chromatin of sperm cells is thought to serve as a mechanism to
366
insure genome integrity, e.g. by suppression of TE transposition because uncontrolled TE
367
transposition could cause insertions, deletions and/or other rearrangements of the genome
368
(Slotkin et al. 2009). Distinct from sperm cells, the pollen vegetative cell does not transfer its
369
genomic DNA to the fertilization products during plant reproduction. The less condensed
370
chromatin allows active TE transcription, and production of mobile siRNAs from TEs in the
371
pollen vegetative cell has been proposed to move to promote TE silencing associated with
372
DNA methylation in the pollen sperm cell nuclei (Ibarra et al. 2012; Slotkin et al. 2009;
373
Calarco et al. 2012). Genome-wide analysis using Arabidopsis seedlings reveals that
374
H3K4me2/3 and DNA methylation are mutually exclusive (Zhang et al. 2009). The sdg2-1
375
mutant has reduced H3K4me3 levels and its pollen vegetative cell exhibits more condensed
376
chromatin (Fig. 4 and 5). Consistent with the role of H3K4me3 in promoting
377
euchromatinization and transcription activation, expression of the retrotransposon
378
ATLANTYS1 and likely also some other TE genes is impaired in the sdg2-1 pollen (Fig. 6, Table
379
S1). Remarkably, the sdg2-1 pollen nuclei display ectopic speckles of H3K9me2, the main
380
histone modification associated with DNA methylation in heterochromatinization in somatic
381
tissues of Arabidopsis (Stroud et al. 2013). It is currently unknown whether or not H3K9me2
382
heterochromatinization is associated with TE silencing and/or whether the H3K4me3
383
reduction directly or indirectly causes the impaired TE expression in the sdg2-1 mutant
384
pollen.
385
In wild-type Arabidopsis pollen, H3K4me3 is found in both the vegetative cell
386
nucleus and the sperm nuclei, whereas H3K9me2 is present specifically in the sperm cell
387
nuclei (Fig. 5; Schoft et al. 2009). Remarkably, a recent study demonstrates that H3K4me3 is
388
present at similar levels in vegetative, generative and sperm nuclear extracts of lily (Lilium
389
davidii) pollen, whereas H3K9me2 and H3K9me3 are detected at higher levels in vegetative
390
than in generative or sperm nuclear extract (Yang et al. 2016). Thus, pollen cell-type-specific
391
pattern of H3K9me2 accumulation is likely plant species-dependent. Nevertheless, H3K9me2
392
in the lily (Lilium longiflorum) pollen vegetative cell nucleus is co-localized with DAPI-staining
393
regions (Sano and Tanaka 2010), indicating that the heterochromatic nature of H3K9me2
394
remains similar in lily as in Arabidopsis. Our study showed that albeit reduced H3K4me3,
395
chromatin compaction remains largely unaffected in the sdg2-1 pollen sperm nuclei. It
396
becomes evident that future studies are required to further investigate histone methylation
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
397
function in chromatin condensation in pollen sperm cells. Distinctively, our data clearly
398
establish a crucial function of H3K4me3 deposition in the pollen vegetative cell. SDG2-
399
dependent H3K4me3 deficiency prevents pollen germination and pollen tube elongation.
400
Chromatin landscape seems to be tightly associated with the active transcriptional genome
401
program of the pollen vegetative cell, which is key for pollen tube growth to guide the
402
delivery of sperm cells to the ovule for fertilization.
403
404
MATERIALS AND METHODS
405
406
Plant material
407
sdg2-1 mutant has been described previously (Berr et al. 2010). Seeds from the reporters
408
AC26-mRFP and HTR10-mRFP were kindly provided by Frederic Berger (Chen et al. 2008).
409
sdg2-1 mutant and the two reporter lines mentioned previously are in the Col-0 background.
410
Both reporter lines were crossed with sdg2-1/+. Plants homozygous for the fluorescent
411
marker and heterozygous for sdg2-1/+ were selected in F2 population by analyzing RFP
412
segregation in mature pollen, and PCR-based genotyping (Berr et al. 2010) to identify sdg2-1
413
allele. Then F3 plants homozygous for both the fluorescent reporter and sdg2-1 were
414
identified based on sdg2-1 phenotype (Berr et al. 2010). The GUS enhancer trap line ET5729
415
was kindly provided by Mathieu Ingouff. ET5729 is in the Landsberg erecta (Ler) background
416
and was crossed to wild-type Col-0 plants (controls) and sdg2-1/+ plants to generate in F3
417
progeny plants homozygous for sdg2-1 and ET5729. Seedlings (until one week old) were
418
grown at 21-22°C under continuous white light and on Murashige and Skoog medium
419
supplemented with 0.8% of Plant agar (Sigma). Then seedlings were transferred on standard
420
potting soil at 21-22°C under a 12h light/12h dark cycle for 3 weeks, and then to a
421
photoperiod of 16h light/8h dark.
422
423
Chromosome spreading analysis and immunostaining
424
Microspores from all stages of meiosis were collected from fresh floral buds, fixed in
425
Carnoy’s fixative solution (ethanol:chloroform:acetic acid = 6:3:1), prepared for chromosome
426
spreads as described previously (Ross et al. 1996), and stained with 4, 6-diamidino-2-
427
phenylindole (DAPI) (0.1 μg/mL). Immunostainings of histone modifications were performed
428
on bicellular and tricellular pollen collected after anther dissection from floral stages 10 to
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
429
12. The collected pollens were fixed in 4% paraformaldehyde in PBS for 30 min, and then
430
keep in 1% sucrose solution overnight at 4°C. After washing, the samples were incubated
431
with the anti-H3K4me3 antibody (Millipore), or the anti-H3K9me2 antibody (Millipore) at
432
1:200 dilution. Alexa Fluor 488-conjugated anti-rabbit IgG antibodies (Invitrogen) and 594-
433
conjugated anti-mouse IgG antibodies (Invitrogen) were used specifically as the secondary
434
antibody.
435
436
Microscopy
437
DAPI staining of pollen grains was performed according to a previous report (Park et al.
438
1998). Epifluoresce images (mRFP-fusion proteins and DAPI-stained DNA) were acquired
439
using Zeiss Imager A2 microscope (Fig. 1 and Fig. 5) and Zeiss LSM710 confocal laser-
440
scanning microscopy (Fig. 2, Fig. 3, Fig. 4, and Fig. 6). GUS staining was performed following
441
the protocol described previously (Johnson et al. 2004). Here pollen grains were incubated
442
with X-Gluc overnight at 37°C, and imaged under a phase-contrast microscope.
443
444
Histone extraction and western blot analysis
445
Histone-enriched proteins were extracted from inflorescence according a previously
446
described method (Yu et al. 2004). Western blot assays were performed to detect the
447
covalent modification status of H3 tails (Yu et al. 2004). Antibodies against histone
448
H3K4me3, H3K9me2, and H3 (Millipore) were used at 1:2000 dilution.
449
450
Quantification and statistic test
451
Densitometry quantification of western blots and fluorescent intensity quantification of
452
microscopy images were performed by using ImageJ software (https://imagej.nih.gov/ij/).
453
For each pollen grain, confocal image with maximum fluorescence intensity was used in
454
measurement at individual nucleus level, divided by nuclear area size, and normalized using
455
a similar size area in the cytoplasm as background. Statistical analysis was performed by
456
unpaired
457
http://www.graphpad.com).
t-test
using
GraphPad
InStat
v.
3.05
(GraphPad
458
459
In vitro pollen germination assay
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Software;
460
In vitro pollen tube growth assay was performed as described previously (Li et al. 1999).
461
Briefly, pollen grains from dehiscent anthers were carefully plated onto the surface of agar
462
plates. The plates were than immediately transferred to a chamber at 25°C and 100%
463
relative humidity. Pollen grains were cultured for 8h, and the germination ratio was
464
calculated by counting 300 pollen grains under a light microscope. This experiment was
465
repeated three times with three plates per repeat.
466
ACKNOWLEDGMENTS
467
The authors thank Zhihao Cheng for technical assistance in chromosome spreading
468
experiments, Hongxing Yang, Yingxiang Wang and Hong Ma for providing male meiocytes
469
transcriptome data and helpful discussions.
470
471
SUPPLEMENTAL DATA
472
Supplemental Figure 1. Immunofluorescence-staining analysis of the wild-type control Col-0
473
and the sdg2-1 mutant pollen.
474
Supplemental Table 1. List of transposable element genes down-regulted by more than 2-
475
fold sdg2-1 as compared to wild-type flower buds.
476
477
FIGURE LEGENDS
478
479
Fig. 1. Male meiosis proceeds normally in the loss-of-function mutant sdg2-1. A to J, DAPI
480
staining showing different stages of meiosis in the wild-type control Col-0. K to T, DAPI
481
staining showing different stages of meiosis in sdg2-1. The shown stages correspond to
482
Zygotene (A, K), Diakinesis (B, L), Metaphase I (C, M), Anaphase I (D, N), Telophase I (E, O),
483
Interphase II (F, P), Metaphase II (G, Q), Anaphase II (H, R), Telophase II (I, S), and newly
484
formed tetrad (J, T). Scale bar = 10 µm for all images (A to T).
485
486
Fig. 2. Male cell fate acquisition is unaffected in the loss-of-function mutant sdg2-1. A and B,
487
DAPI (upper panels) and HTR10-mRFP (lower panels) fluorescent signals in pollen at
488
bicellular and tricellular developmental stage, respectively. In (B) but not in (A), the HTR10-
489
mRFP fluorescence is shown together with its corresponding transmitted light reference
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
490
image to visualize the entire mature pollen grain. Note for specific HTR10-mRFP expression
491
in the generative (A) and sperm (B) cells within pollen grains in sdg2-1 similarly as in the
492
wild-type control Col-0. C, DAPI (upper panels) and AC26-mRFP (lower panels) fluorescent
493
signals in tricellular stage pollen in Col-0 and sdg2-1. Note for specific AC26-mRFP signal
494
detected in the vegetative cell nucleus but not in the sperm cells, similarly in sdg2-1 and Col-
495
0. Scale bars = 10 µm for all images.
496
497
Fig. 3. Loss of SDG2 prevents mitotic division of pollen generative cell in the sdg2-1 mutant.
498
A, Graphic representation of pollen grains containing the indicated number of generative cell
499
(GC) determined using HTR10-mRFP marker in sdg2-1 and the wild-type control Col-0 at
500
bicellular pollen developmental stage. Pollen was examined from anthers at developmental
501
stage 11 from over ten individual plants for each genotype. The difference between sdg2-1
502
and Col-0 is not significant according to unpaired t-test (p-value > 0.05). B, Graphic
503
representation of pollen grains containing the indicated number of sperm cell (SC)
504
determined using HTR10-mRFP marker in sdg2-1 and Col-0 at tricellular pollen
505
developmental stage. Pollen was examined from anthers at developmental stage 12-13 from
506
over ten individual plants for each genotype. Unpaired t-test indicates that the difference
507
between sdg2-1 and Col-0 is statistically significant for 1 SC and 2 SC categories (indicated by
508
*, p-values < 0.05) but not for 0 SC category (p-value > 0.05). C, HTR10-mRFP (left panel) and
509
DAPI (right panel) fluorescent signals in sdg2-1 pollen grains at tricellular pollen
510
developmental stage. Pollen was examined from anthers at developmental stage 12-13.
511
Arrows indicate pollen grains arrested at bicellular stage without GC division. Scale bars = 10
512
µm.
513
514
Fig. 4. SDG2 is involved in promoting chromatin decondensation in microspore and in pollen
515
vegetative cell nuclei. A, DAPI fluorescent signals in microspores and in pollen grains at
516
bicellular or tricellular stage in the wild-type control Col-0 and the loss-of-function mutant
517
sdg2-1, as indicated. Note for more condensed chromatin in sdg2-1 than in Col-0 in
518
microspore and in the vegetative cell of bicellular or tricellular pollen. Dashed arrow
519
indicates the vegetative cell nucleus and solid arrows indicate sperm cells in the tricelluar
520
pollen. Scale bars = 10 µm. B, Graphic representation of the nuclei area from sperm cells and
521
vegetative cells of mature tricellular stage pollen. The area was measured using ImageJ
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
522
software from DAPI-staining microscopy images at the largest scan surface. C, Graphic
523
representation of the DAPI fluorescence intensity per µm2 from sperm and vegetative cell
524
nuclei of mature tricellular stage pollen. The fluorescence intensity was measured using
525
ImageJ software from the image scan displaying the brightest signal. Data shown in B and C
526
represent mean together with error bar based on 40 pollen grains for each genotype per
527
experiment from three independent experiments. The symbol * indicates where the
528
difference between Col-0 and sdg2-1 is significant in statistic test (p-value < 0.05).
529
530
Fig. 5. SDG2 contributes to H3K4me3 and H3K9me2 patterning in pollen. A,
531
Immunofluorescence staining performed with anti-H3K4me3 antibody in tricellular pollen in
532
the wild-type control Col-0 (upper panels) and the sdg2-1 mutant (lower panels). DAPI
533
staining of the same pollen is shown on left panel. Note for drastically reduced H3K4me3
534
signal in the vegetative cell nucleus and the two sperm nuclei of the sdg2-1 pollen as
535
compared to the Col-0 pollen. B, Graphic representation of the relative H3K4me3
536
fluorescence intensity, after normalization using DAPI, in sperm cell and vegetative cell
537
nuclei of Col-0 and sdg2-1 (set as 1 for sdg2-1 vegetative cell nuclei). The fluorescence
538
intensity was measured using ImageJ software, and mean values from 10 pollen grains are
539
shown together with error bars. The symbol * indicates where the difference between Col-0
540
and sdg2-1 is significant in statistic test (p-value < 0.05). C, Western blot analysis of
541
H3K4me3 and H3K9me2 levels in sdg2-1. Histone-enriched protein were extracted from
542
inflorescence of Col-0 and sdg2-1 plants grown under a 16h light / 8h dark photoperiod.
543
H3K9me2, H3K4me3 and total H3 proteins were detected using specific antibodies. Numbers
544
indicate 'mean (± standard error)' of relative densitometry values measured from 3
545
independent experiments, in sdg2-1 and Col-0 (set as 1). D, Immunofluorescence staining
546
performed with anti-H3K9me2 antibody in tricellular pollen in Col-0 (upper panels) and
547
sdg2-1 (lower panels). DAPI staining of the same pollen is shown on left panel. Note for
548
H3K9me2 speckles detected in the sdg2-1 pollen vegetative cell nucleus but not in the Col-0
549
pollen vegetative cell nucleus. E, Close-up images showing H3K9me2 speckles in the sdg2-1
550
but not in the Col-0 pollen vegetative nucleus (dashed circle) as well as in the sperm nuclei
551
(circles) of both sdg2-1 and Col-0 pollen. F, Images showing H3K9me2 speckles in the
552
vegetative nucleus (dashed circle) and in the generative nucleus (circles) of bicellular pollen
553
in Col-0 and sdg2-1. Scale bars = 10 µm for A and D, and 5 µm for E and F.
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
554
555
Fig. 6. SDG2 is involved in activation of retransposon ATLANTYS1 transcription and in
556
promoting pollen germination and pollen tube growth. A-C, GUS staining of tricellular stage
557
pollen in bright field microscopy from Col-0 (A), from a F2 plant generated by the cross
558
between the wild-type control Col-0 and the enhancer trap line ET5729 (B), or between
559
sdg2-1 and ET5729 (C). D and E, GUS staining of tricellular stage pollen from plants
560
homozygous for ET5729 in respectively Col-0 (D) and sdg2-1 (E) backgrounds. The lower
561
panels show the corresponding DAPI stained pollen. Arrows indicate the sperm cell nuclei. F,
562
Representative images showing in vitro germination of pollen from Col-0 and sdg2-1. Red
563
arrows indicate germinated sdg2-1 pollen but with pollen tube elongation inhibited. G,
564
Graphic representation of sdg2-1 and Col-0 pollen germination rate. Scale bars = 10 µm for
565
A-E, and 200 µm for F.
566
567
568
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Col-0
Col-0
Col-0
Col-0
Col-0
Col-0
Col-0
Col-0
Col-0
Col-0
sdg2-1
sdg2-1
sdg2-1
sdg2-1
sdg2-1
sdg2-1
sdg2-1
sdg2-1
sdg2-1
sdg2-1
Fig. 1. Male meiosis proceeds normally in the loss-of-function mutant sdg2-1. A to J, DAPI
staining showing different stages of meiosis in the wild-type control Col-0. K to T, DAPI
staining showing different stages of meiosis in sdg2-1. The shown stages correspond to
Zygotene(A,K),Diakinesis(B,L),MetaphaseI(C,M),AnaphaseI(D,N),TelophaseI(E,O),
Interphase II (F, P), Metaphase II (G, Q), Anaphase II (H, R), Telophase II (I, S), and newly
formedtetrad(J,T).Scalebar=10µmforallimages(AtoT).
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
A
Col-0
B
sdg2-1
Col-0
sdg2-1
HTR10-mRFP
HTR10-mRFP
C
Col-0
sdg2-1
AC26-mRFP
Fig.2.Malecellfateacquisitionisunaffectedintheloss-of-functionmutantsdg2-1.AandB,
DAPI (upper panels) and HTR10-mRFP (lower panels) fluorescent signals in pollen at
bicellularandtricellulardevelopmentalstage,respectively.In(B)butnotin(A),theHTR10mRFP fluorescence is shown together with its corresponding transmitted light reference
imagetovisualizetheentirematurepollengrain.NoteforspecificHTR10-mRFPexpression
in the generative (A) and sperm (B) cells within pollen grains in sdg2-1 similarly as in the
wild-type control Col-0. C, DAPI (upper panels) and AC26-mRFP (lower panels) fluorescent
signals in tricellular stage pollen in Col-0 and sdg2-1. Note for specific AC26-mRFP signal
detectedinthevegetativecellnucleusbutnotinthespermcells,similarlyinsdg2-1andCol0.Scalebars=10µmforallimages.
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
A
98%
94%
(n=284)
C
B
Bicellularstage
Col
sdg2-1
Col-0
sdg2-1
2%
6%
(n=266)
1GC
0GC
Tricellularstage
Col
sdg2-1
sdg2-1
Col-0
8%
11%
11%
24%*
82%
(n=311)
65%*
2SC
1SC
0SC
(n=325)
Tricellularstage
HTR10-mRFP
DAPI
Fig.3.LossofSDG2preventsmitoticdivisionofpollengenerativecellinthesdg2-1mutant.
A,Graphicrepresentationofpollengrainscontainingtheindicatednumberofgenerativecell
(GC) determined using HTR10-mRFP marker in sdg2-1 and the wild-type control Col-0 at
bicellularpollendevelopmentalstage.Pollenwasexaminedfromanthersatdevelopmental
stage11fromovertenindividualplantsforeachgenotype.Thedifferencebetweensdg2-1
and Col-0 is not significant according to unpaired t-test (p-value > 0.05). B, Graphic
representation of pollen grains containing the indicated number of sperm cell (SC)
determined using HTR10-mRFP marker in sdg2-1 and Col-0 at tricellular pollen
developmentalstage.Pollenwasexaminedfromanthersatdevelopmentalstage12-13from
over ten individual plants for each genotype. Unpaired t-test indicates that the difference
betweensdg2-1andCol-0isstatisticallysignificantfor1SCand2SCcategories(indicatedby
*,p-values<0.05)butnotfor0SCcategory(p-value>0.05).C,HTR10-mRFP(leftpanel)and
DAPI (right panel) fluorescent signals in sdg2-1 pollen grains at tricellular pollen
developmental stage. Pollen was examined from anthers at developmental stage 12-13.
ArrowsindicatepollengrainsarrestedatbicellularstagewithoutGCdivision.Scalebars=10
µm.
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
sdg2-1
Fluorescence Intensity / µm2
Nucleiarea(µm2)
Col-0
10000"
C
C
10000"
Sperm"cell"nuclei"
Vegeta;ve"cell"nuclei"
8000"
Microspore
6000"
Bicellularstage
4000"
*
*
2000"
Tricellularstage
0"
Col-0
Col"
sdg2-1
sdg2.1"
sdg21
2 2
FluorescenceIntensity/µm
Fluorescence
Intensity / µm
C
B
A
Sperm"cell"nuclei"
Vegeta;ve"cell"nuclei"
8000"
6000"
4000"
*
*
2000"
0"
Col-0
Col"
sdg2-1
sdg2.1"
sdg21
Fig.4.SDG2isinvolvedinpromotingchromatindecondensationinmicrosporeandinpollen
vegetative cell nuclei. A, DAPI fluorescent signals in microspores and in pollen grains at
bicellular or tricellular stage in the wild-type control Col-0 and the loss-of-function mutant
sdg2-1, as indicated. Note for more condensed chromatin in sdg2-1 than in Col-0 in
microspore and in the vegetative cell of bicellular or tricellular pollen. Dashed arrow
indicates the vegetative cell nucleus and solid arrows indicate sperm cells in the tricelluar
pollen.Scalebars=10µm.B,Graphicrepresentationofthenucleiareafromspermcellsand
vegetative cells of mature tricellular stage pollen. The area was measured using ImageJ
software from DAPI-staining microscopy images at the largest scan surface. C, Graphic
representation of the DAPI fluorescence intensity per µm2 from sperm and vegetative cell
nuclei of mature tricellular stage pollen. The fluorescence intensity was measured using
ImageJsoftwarefromtheimagescandisplayingthebrightestsignal.DatashowninBandC
represent mean together with error bar based on 40 pollen grains for each genotype per
experiment from three independent experiments. The symbol * indicates where the
differencebetweenCol-0andsdg2-1issignificantinstatistictest(p-value<0.05).
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
DAPI
H3K4me3
B
Col-0
Vegeta=vecellnuclei
D
DAPI
Col-0 sdg2-1
H3
1
0.80±0.10
1
0.95±0.09
1
0.21±0.06
*
H3K9me2
*
Col-0
E
sdg2-1
C
Spermcellnuclei
Rela=veintensity
A
H3K9me2
sdg2-1
F
H3K4me3
H3K9me2
H3K9me2
Col-0
Col-0
Col-0
sdg2-1
sdg2-1
sdg2-1
Fig. 5. SDG2 contributes to H3K4me3 and H3K9me2 patterning in pollen. A,
Immunofluorescencestainingperformedwithanti-H3K4me3antibodyintricellularpollenin
the wild-type control Col-0 (upper panels) and the sdg2-1 mutant (lower panels). DAPI
staining of the same pollen is shown on left panel. Note for drastically reduced H3K4me3
signal in the vegetative cell nucleus and the two sperm nuclei of the sdg2-1 pollen as
compared to the Col-0 pollen. B, Graphic representation of the relative H3K4me3
fluorescence intensity, after normalization using DAPI, in sperm cell and vegetative cell
nuclei of Col-0 and sdg2-1 (set as 1 for sdg2-1 vegetative cell nuclei). The fluorescence
intensitywasmeasuredusingImageJsoftware,andmeanvaluesfrom10pollengrainsare
showntogetherwitherrorbars.Thesymbol*indicateswherethedifferencebetweenCol-0
and sdg2-1 is significant in statistic test (p-value < 0.05). C, Western blot analysis of
H3K4me3 and H3K9me2 levels in sdg2-1. Histone-enriched protein were extracted from
inflorescence of Col-0 and sdg2-1 plants grown under a 16h light / 8h dark photoperiod.
H3K9me2,H3K4me3andtotalH3proteinsweredetectedusingspecificantibodies.Numbers
indicate 'mean (± standard error)' of relative densitometry values measured from 3
independent experiments, in sdg2-1 and Col-0 (set as 1). D, Immunofluorescence staining
performed with anti-H3K9me2 antibody in tricellular pollen in Col-0 (upper panels) and
sdg2-1 (lower panels). DAPI staining of the same pollen is shown on left panel. Note for
H3K9me2specklesdetectedinthesdg2-1pollenvegetativecellnucleusbutnotintheCol-0
pollenvegetativecellnucleus.E,Close-upimagesshowingH3K9me2specklesinthesdg2-1
butnotintheCol-0pollenvegetativenucleus(dashedcircle)aswellasinthespermnuclei
(circles) of both sdg2-1 and Col-0 pollen. F, Images showing H3K9me2 speckles in the
vegetativenucleus(dashedcircle)andinthegenerativenucleus(circles)ofbicellularpollen
inCol-0andsdg2-1.Scalebars=10µmforAandD,and5µmforEandF.
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
A
Col-0
Col-0
xET5729
E
D
C
B
sdg2-1
xET5729
ET5729(Col-0)
ET5729(sdg2-1)
G
Germina9onrate(%)
F
Col-0
(n=300)
(n=300)
DAPI
DAPI
Col-0
sdg2-1
Fig. 6. SDG2 is involved in activation of retransposon ATLANTYS1 transcription and in
promotingpollengerminationandpollentubegrowth.A-C,GUSstainingoftricellularstage
pollen in bright field microscopy from Col-0 (A), from a F2 plant generated by the cross
between the wild-type control Col-0 and the enhancer trap line ET5729 (B), or between
sdg2-1 and ET5729 (C). D and E, GUS staining of tricellular stage pollen from plants
homozygous for ET5729 in respectively Col-0 (D) and sdg2-1 (E) backgrounds. The lower
panelsshowthecorrespondingDAPIstainedpollen.Arrowsindicatethespermcellnuclei.F,
Representative images showing in vitro germination of pollen from Col-0 and sdg2-1. Red
arrows indicate germinated sdg2-1 pollen but with pollen tube elongation inhibited. G,
Graphicrepresentationofsdg2-1andCol-0pollengerminationrate.Scalebars=10µmfor
A-E,and200µmforF.
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