Plant Physiology Preview. Published on October 8, 2015, as DOI:10.1104/pp.15.00861
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Running head: Chlamydomonas life cycle transcriptome and methylome
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Corresponding author:
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Matteo Pellegrini
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Department of Molecular, Cell, and Developmental Biology
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University of California, Los Angeles
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Los Angeles, California, 90095, USA
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Tel: (310) 825-0012
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[email protected]
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Research area: Genes, Development, and Evolution
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Secondary research area: Cell Biology
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Copyright 2015 by the American Society of Plant Biologists
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Dynamic changes in the transcriptome and methylome of Chlamydomonas
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reinhardtii throughout its life cycle
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David Lopez1,2, Takashi Hamaji3, Janette Kropat4, Peter De Hoff5,6, Marco Morselli2,
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Liudmilla Rubbi2, Sorel Fitz-Gibbon2,4, Sean D. Gallaher4, Sabeeha S. Merchant4,7, James
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Umen3, Matteo Pellegrini2,7
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Molecular Biology Institute, University of California, Los Angeles, Los Angeles,
California
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Department of Molecular, Cell, and Developmental Biology, University of California,
Los Angeles, Los Angeles, California
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Donald Danforth Plant Science Center, St. Louis, Missouri
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Department of Chemistry and Biochemistry, University of California, Los Angeles, Los
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Angeles, California
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Salk Institute for Biological Studies, La Jolla, California
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Current address: Synthetic Genomics, La Jolla, California
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Institute for Genomics and Proteomics, University of California, Los Angeles, Los
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Angeles, California
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One-sentence summary:
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Secretory and algal-specific genes are induced during gamete and zygote development in
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C. reinhardii, concurrent with a dramatic increase in chloroplast cytosine methylation.
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Funding was provided by the National Institutes of Health R24 GM092473, R01
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GM078376, and by the Office of Science (Biological and Environmental Research), U.S.
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Department of Energy (grant no. DE–FC02–02ER63421). David Lopez is supported by
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an NIH T32 Training Fellowship in Genome Analysis (5T32HG002536-13), the Eugene
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V. Cota-Robles Fellowship, and the Fred Eiserling and Judith Lengyel Doctoral
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Fellowship. Takashi Hamaji is a JSPS Postdoctral Fellow for Research Abroad (26-495)
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of the Japan Society for the Promotion of Science.
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Author Contributions
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MP, JU, and SM designed the experiment. JK, MM, LR, and PDH collected the samples
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and created the sequence libraries in preparation for sequencing. DL and TH aligned the
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sequence data and performed all bioinformatic analyses, with the exception of the
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analysis of the genomic data and identification of structural variants, which was carried
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out by SFG and SG. The manuscript was written by DL, JU, MP, and SM.
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Abstract
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The green alga Chlamydomonas reinhardtii undergoes gametogenesis and mating upon
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nitrogen starvation. While the steps involved in its sexual reproductive cycle have been
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extensively characterized, the genome-wide transcriptional and epigenetic changes
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underlying different life cycle stages have yet to be fully described. Here, we performed
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transcriptome and methylome sequencing to quantify expression and DNA methylation
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from vegetative and gametic cells of each mating-type and from zygotes. We identified
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361 gametic genes with mating-type-specific expression patterns, and 627 genes that are
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specifically induced in zygotes; furthermore, these sex-related gene sets were enriched
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for secretory pathway and algal-specific genes. We also examined the C. reinhardtii
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nuclear methylation map with base-level resolution at different life cycle stages. Despite
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having low global levels of nuclear methylation, we detected 23 hypermethylated loci in
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gene-poor, repeat-rich regions. We observed mating-type-specific differences in
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chloroplast DNA methylation levels in plus versus minus mating type gametes followed
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by chloroplast DNA hypermethylation in zygotes. Lastly, we examined expression of
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candidate DNA methyltransferases and found three – DMT1a, DMT1b and DMT4 – that
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are differentially expressed during the life cycle and are candidate DNA methylases. The
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expression and methylation data we present provide insight into cell-type-specific
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transcriptional and epigenetic programs during key stages of the C. reinhardtii life cycle.
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Introduction
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Chlamydomonas reinhardtii is a unicellular, biflagellate species of green alga found
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primarily in fresh water and soil (Harris et al., 2009). C. reinhardtii is an important
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reference organism for diverse eukaryotic cellular and metabolic processes including
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photosynthetic biology (Rochaix, 2001), flagellar function and biogenesis (Silflow and
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Lefebvre, 2001), nutrient homeostasis (Grossman, 2000; Merchant et al., 2006; Glaesener
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et al., 2013) and sexual cycles (Goodenough et al., 2007). The nuclear and chloroplast
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genomes of C. reinhardtii have been fully sequenced, enabling genomic and epigenomic
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analyses (Maul et al., 2002; Merchant et al., 2007). The ~112 Mbp haploid C. reinhardtii
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nuclear genome comprises 17 chromosomes. The circular chloroplast genome (cpDNA)
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is 203 kbp and present in 80-100 copies per cell that are organized into 8-10
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nucleoprotein complexes called nucleoids, which are distributed through the stroma.
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Like many unicellular eukaryotes, C. reinhardtii has a biphasic life cycle where haploid
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cells can reproduce vegetatively by mitotic division or, alternatively, undergo a sexual
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cycle. Vegetative cells can propagate indefinitely when provided with nutrients and light.
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Upon nitrogen starvation, however, cells stop dividing and differentiate into gametes
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whose mating type (plus or minus) is determined genetically by a ~300 kb mating-type
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locus (MT) on chromosome 6, with two haplotypes, MT+ and MT− (Umen, 2011; De
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Hoff et al., 2013). Gametes express a set of mating-related proteins that are different
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between minus and plus cells and which allow cells of opposite mating-type to recognize
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each other and fuse to form a quadriflagellate zygote. Upon fertilization, the
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heterodimeric KNOX/BELL-type homeodomain proteins GSM1 and GSP1 initiate a
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zygote-specific developmental program which includes flagellar resorption, fusion of
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organelles including nuclei and chloroplasts, destruction of MT− cpDNA, and secretion
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of a thick, environmentally-resistant cell wall that protects the zygospore from cold,
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dessication and other environmental stresses (Cavalier-Smith, 1976; Catt, 1979; Grief et
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al., 1987; Brawley and Johnson, 1992; Goodenough et al., 2007; Lee et al., 2008). Upon
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return to favorable conditions of light and nutrients, zygospores undergo meiosis to
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produce four haploid progeny (2 MT+, 2 MT−) that can reenter the vegetative life cycle.
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While nuclear loci segregate in a Mendelian pattern of 2:2, both chloroplast and
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mitochondrial genomes are inherited uniparentally, with cpDNA inherited from the MT+
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parent and mitochondrial DNA (mtDNA) from the MT− parent (Nakamura, 2010;
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Nishimura, 2010).
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While previous high-throughput expression studies have focused on the transcriptional
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programs underlying processes such as nutrient deprivation (Nguyen et al., 2008;
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Gonzalez-Ballester et al., 2010; Toepel et al., 2011; Toepel et al., 2013; Schmollinger et
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al., 2014), environmental responses (Simon et al., 2008; Matsuo et al., 2011; Fang et al.,
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2012; Simon et al., 2013), flagellar biogenesis (Albee et al., 2013), lipid accumulation
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(Miller et al., 2010; Boyle et al., 2012; Lv et al., 2013), and diurnal rhythms (Idoine et al.,
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2014; Panchy et al., 2014), only a few studies have explored the genome-wide
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transcriptional and epigenetic changes associated with the sexual cycle (Kubo et al.,
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2008; Ning et al., 2013; Aoyama et al., 2014). Several genes expressed in the early
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zygote, termed EZY genes, have predicted functions related to cell wall production,
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vesicular transport, and secretion (Ferris and Goodenough, 1987; Ferris et al., 2002;
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Kubo et al., 2008). A separate analysis of zygospore transcripts following light-induced
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germination revealed the upregulation of photosynthetic and methionine synthesis
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pathways (Aoyama et al., 2014).
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DNA methylation studies have also been conducted on both the nuclear and chloroplast
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genomes (Hattman et al., 1978; Dyer, 1982). cpDNA methylation has been studied more
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extensively and shows dramatic changes in 5meC content at different stages of the C.
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reinhardtii life cycle. Vegetative cells have low levels of 5meC in cpDNA, while
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gametes show a substantial increase with cpDNA (12% 5meC in MT+ gamete cells and
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4% in MT–) (Royer and Sager, 1979; Feng and Chiang, 1984). In zygotes, MT− cpDNA
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is eliminated while MT+ cpDNA becomes hypermethylated. While differential cpDNA
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methylation was once thought to be part of a restriction-methylation system regulating
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uniparental inheritance (Burton et al., 1979), this model is unlikely since loss of cpDNA
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methylation in MT+ cells does not result in its destruction in zygotes (Umen and
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Goodenough, 2001), and ectopic methylation of MT− cpDNA does not spare it from
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destruction (Bolen et al., 1982). However, previous studies are consistent with a role for
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5meC in promoting cpDNA replication upon zygote germination, which can influence the
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amount of residual MT− cpDNA that is inherited by exceptional progeny (Umen and
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Goodenough, 2001; Nishiyama et al., 2004). An alternative proposed mechanism
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involves digestion of MT− cpDNA by differentially-localized or -activated nucleases that
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are methylation-insensitive early in zygote development before chloroplast fusion
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(Nishimura et al., 2002).
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Several methyltransferase enzymes that modify chloroplast DNA have been investigated
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biochemically (Sano et al., 1981) and one candidate chloroplast methyltransfersase gene
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has been cloned (Nishiyama et al., 2002; Nishiyama et al., 2004). Since this time, the
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genome sequence of Chlamydomonas reinhardtii has become available (Merchant et al.,
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2007) and extensively annotated (Blaby et al., 2014) so that a comprehensive
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identification of genes encoding DNA methyltransferases can be undertaken.
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Few studies have focused on the role of nuclear cytosine methylation in Chlamydomonas,
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but previous work has shown that induced silencing of nuclear transgenes does not
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correlate with transgene cytosine methylation levels, leaving open the question of what
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role cytosine methylation plays in chromatin structure and gene expression in C.
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reinhardtii (Cerutti et al., 1997). To date, the absence of detailed methylation maps has
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precluded a clear view of methylation patterns in the nuclear and chloroplast genomes of
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Chlamydomonas.
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Here we have performed RNA-seq based transcriptome analysis and bisulfite-DNA
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sequencing of C. reinhardtii at different life cycle stages. We identify sex- and mating-
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related changes in gene expression including genes that are preferentially expressed in
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gametes of each mating type or in zygotes. We generated a high-resolution map of
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nuclear and chloroplast cytosine methylation during the life cycle, and identified
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candidate DNA methyltransferases whose expression profiles correlate with dynamic
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changes in cpDNA methylation patterns.
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Results
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To quantify nuclear gene expression and 5-cytosine DNA methylation throughout the C.
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reinhardtii life cycle, we collected RNA and DNA samples at various stages for RNA
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and whole genome bisulfite (WGBS) sequencing (Figure 1). Samples were collected
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from vegetative and gametic cells of both mating types and zygotic stages to enable
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multiple comparisons. The design of the experiment was for matched DNA and RNA
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samples, but the RNA protocol was modified to avoid degradation. We note that for
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transcriptome studies, our vegetative samples were prepared in a different manner than
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those used for typical N-starvation studies. We grew all cultures to saturation on solid
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agar media and then resuspended cells at high density (~2 x 107 mL-1) under illumination
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in liquid medium (HSM) with (+N) or without (−N) nitrogen for ~3-5 hours. Under these
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conditions neither culture grew measurably, but the –N cultures expressed the gametic
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program and efficiently mated at >90% efficiency; while the +N cultures could not mate
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and did not express gametic marker genes (see below). The advantage of this procedure
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is that it minimizes the differences between +N and –N cultures that would normally be
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attributable to growth rate differences and thereby allows more reliable identification of
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mating-related gene expression. For DNA methylation studies, the vegetative and
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gametic samples were obtained from growing N-replete and N-starved samples
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respectively as described in Materials and Methods. Actively growing cultures were used
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for studies of methylation in vegetative cells since agar plate grown cells are already
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partially gametic and would require additional rounds of division in order to remove
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preexisting methylation.
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Sequence polymorphisms between R3 (MT+) and CJU10 (MT–) parental strains
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As a prelude to transcriptome and methylome analyses, we cataloged the genetic
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differences (single nucleotide variants, insertions, and deletions) between our two
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parental strains using genome resequencing (Figure 2A) (Gallaher et al., 2015). In total,
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the two strains differ by 0.16% in their nuclear genomes, and most of these differences
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are single nucleotide variants (SNVs). Of all the variants, 98.2% are localized to two
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regions, one of length 2.2 Mb on chromosome 17, and one of 2 Mb encompassing the
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mating locus on chromosome 6 (Figure 2B) where mating-type haplotype differences
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have previously been observed (De Hoff et al., 2013). Additionally, the chloroplast
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genome of the CJU10 strain contains an insertion adjacent to the atpB gene of a
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spectinomycin resistance marker (aadA), flanked by the rbcL 5’ promoter and psbC 3’
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UTR (Figure 2C) (Goldschmidt-Clermont, 1991; Umen and Goodenough, 2001).
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Gamete and zygote-specific genes
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Following the quantitation of RNA-seq data for all of our samples, we identified genes
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with mating type and zygote-specific expression patterns using a series of filters to screen
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for genes matching expression patterns of known gametic and zygotic genes. We
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required that mating-type-specific genes be expressed in gametes at least four-fold higher
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than in vegetative cells of the same mating type, and at least ten fold higher than in
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gametes or vegetative cells of the opposite mating type. For zygote-specific genes, we
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required that expression be at least four-fold higher in early zygotes than in any other
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sample. Using these criteria, we identified 293 and 68 genes whose expression is specific
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to plus and to minus gametes respectively, and 627 genes whose expression is specific to
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zygotes (Figure 3A, Supplementary Table 1). Genes whose expression is known to be
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gamete- or zygote-specific, including GSM1 (minus gametes), SAG1 (plus gametes) and
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EZY1 (early zygotes) were retained by our filters (Armbrust et al., 1993; Kurvari et al.,
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1998; Ferris et al., 2002; Ferris et al., 2005; Lee et al., 2008). Of the 32 previously
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described zygotic genes (Matters and Goodenough, 1992; Armbrust et al., 1993; Uchida
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et al., 1993; Kuriyama et al., 1999; Uchida et al., 1999; Suzuki et al., 2000; Ferris et al.,
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2002; Kubo et al., 2008), 25 were in our zygote data set, while the remaining 7 (EZY6,
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EZY15, EZY16, EZY17, EZY21, EZY23, and LYZ1A) were found to have significant
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expression in other samples and were therefore excluded from the zygote-specific set
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(Supplementary Tables 1 and 2).
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We functionally classified the predicted proteins encoded by genes whose expression was
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plus-gamete-specific, minus-gamete-specific, or early-zygote-specific. Protein
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localization prediction revealed the enrichment of putative secretory proteins in plus-
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gamete (MT+) and zygote-specific sets (Figure 3B). We also assessed conservation and
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phylogenetic distribution for homologs of each protein in a set of nested phylogenetic
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domains that encompass different taxonomic levels from cellular organisms (prokarya,
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archaea and eukarya) to the single species level of C. reinhardtii (Figure 3C). Zygote
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and gamete expression groups were enriched for Chlamydomonas-specific genes and/or
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algal specific genes. In addition, gametic genes were under-represented for more widely
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conserved genes (i.e. those with homologs outside of chlorophyte algae). Consistent with
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these findings, the gametic and zygotic gene lists were also depleted to various extents
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for GreenCut2 and CiliaCut genes whose members are associated with conserved
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photosynthetic and flagella/basal body functions (Merchant et al., 2007; Karpowicz et al.,
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2011; Heinnickel and Grossman, 2013) (Figures 4A and 4B); however, the overall low
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numbers of genes in these categories precluded obtaining a significant statistical result in
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all cases but one. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes
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(KEGG), and MapMan (Kanehisa and Goto, 2000; Harris et al., 2004; Thimm et al.,
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2004) classification of predicted proteins encoded by gametic and zygotic genes was
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performed using the Algal Functional Annotation Tool (Lopez et al., 2011), but had
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limited utility because of the large number of non-conserved proteins in these groups and
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incomplete annotations. Nonetheless, we found significant enrichment in two MapMan
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categories for zygotic genes, “Cell Wall” and “Transport”, both of which may relate to
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the requirement for new cell wall biosynthesis in zygotes (Figures 4C and 4D). Indeed,
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examination of manually curated early zygotic gene annotations revealed numerous cell-
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wall related protein coding genes as described below.
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Volvocine cell walls are composed primarily of glycosylated hydroxyproline-rich
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glycoproteins (HRGPs) that enter the secretory pathway and are exported to the
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extracellular space where they co-assemble (Woessner et al., 1994). The thick and
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environmentally-resistant cell walls of zygospores are formed by a specialized set of
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HRGPs that are synthesized shortly after fertilization (Minami and Goodenough, 1978;
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Catt, 1979; Grief et al., 1987). Manual annotation and inspection of zygotic up-regulated
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genes verified the MapMan ontology assignments of cell wall and transport categories as
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described in Supplementary Table 1. At least 57 zygotic genes are predicted HRGPs or
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have putative cell wall biogenesis-related functions that include secretion, glycosylation
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and metabolism of nucleotide sugars (e.g. UDP-glucose 4-epimerase, pyrophosphorylase,
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dehydrogenase; dTDP-6-deozy-L-lyxo-4-hexulose reductase related, exotosin-like
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glycosyltransferase), or sugar metabolite transport (e.g. ABC transporter, triose
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phosphate transporter, UDP-N-acetylglucosamine transporter). Among these were some
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previously identified early zygotic genes as noted in Supplementary Table 1 (EZY4,
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EZY11/UPT1/UPTG1, EZY12/UGD1, EZY14/TPT14, EZY22) (Kubo et al., 2008).
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Besides cell wall and secretory pathway genes, we also noted in the zygote gene set
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predicted functions that may be related to other zygotic processes including elimination
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of MT– chloroplast DNA, zygotic cpDNA methylation and packaging for long-term
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dormancy, nuclear fusion (karyogamy), chloroplast fusion, and flagellar resorption
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(Goodenough et al., 2007). These annotations include predicted chloroplast-targeted
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DNA binding proteins such as a recA homolog and predicted nuclease
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(EZY19/Cre07.g314650), chloroplast targeted DNA methyltransferases (DMT1A,
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DMT1B, DMT4; discussed below), and a chloroplast-targeted dynamin
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(EZY8/Cre06.g25065) that may be involved in chloroplast fusion (Kubo et al., 2008).
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Predicted nuclear-targeted zygotic proteins include several DNA binding transcription
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factors (EZY18/Cre02.g091550, RLS7/Cre14.g617200, ZYS1A/Cre17.g719200) such as
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RLS7 that contains a SAND-domain (Duncan et al., 2006; Duncan et al., 2007) related to
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RegA, a repressor of germ cell fate in Volvox carteri (Kirk et al., 1999), and several types
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of chromatin-related proteins (Cre03.g184900, Cre08.g367000, Cre08.g400200,
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Cre09.g401812, HON1/Cre13.g567450) that may be involved in nuclear DNA packaging
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in preparation for zygospore dormancy. Minutes after fertilization and prior to flagellar
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resorption the four basal bodies and flagella of newly formed zygotes (two from each
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parent) move to a single apical location via an unknown mechanism. One possible
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participant in this process could be the striated fiber protein SF-assemblin (SFA1/
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Cre07.g332950), a zygote up-regulated gene whose protein product in vegetative cells
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associates with rootlet microtubules that are proximal to basal bodies and are thought to
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play a role in organization of the rootlet structure (Lechtreck et al., 2002). Two other
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cytoskeletal proteins whose genes are up-regulated in zygotes are a flagella-associated
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protein of unknown function, FAP79 (Cre04.g217908) and the flagella length regulatory
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protein LF5/FAP279 (Tam et al., 2013) (Cre12.g538300) which may be related to
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flagellar resorption that begins two or three hours after fertilization. Lastly, we identified
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a gene for a predicted secreted trypsin-related protease (Cre06.g287750), which could
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contribute to rapid post-fertilization degradation of gametic plasma membrane surface
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proteins such as FUS1 and GCS1/HAP2, a process that is thought to restrict polygamy
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(fusion between >2 gametes) (Liu et al., 2010).
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DNA methylation of the nuclear genome
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We conducted bisulfite sequencing of vegetative, gametic and zygotic samples to
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generate DNA methylation profiles. The nuclear genome had an average per-site CG
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methylation of < 0.75% in all samples, and this level of methylation did not differ
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significantly between plus and minus strains or at different life cycle stages (Figure 5A).
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However, CG methylation densities greater than 80% were identified for 23 loci that
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ranged in size between 10 kb and 22 kb (Figure 5B, Supplementary Table 3). The highly-
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methylated regions are enriched for repeats, and their overall protein coding gene
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densities are significantly lower than average, though there are still genes in these regions
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(Figure 5C). In addition, one example where methylation was strain-specific is shown in
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Figure 5D, although most hypermethylated sites did not have strain specific methylation
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patterns. The expression levels of genes overlapping hypermethylated loci are not
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strongly correlated with degree of methylation (Supplementary Table 4).
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Chloroplast methylation changes during the life cycle
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In contrast to the relatively stable pattern of cytosine methylation in nuclear DNA, the C.
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reinhardtii chloroplast genome underwent dynamic changes in cytosine methylation
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throughout the life cycle (Figure 6A). In the vegetative stage, global per-cytosine
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methylation was < 2% for both mating types for all cytosine contexts (CG, CHG, CHH).
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After gametogenesis, cpDNA methylation increased in a mating-type dependent manner.
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MT+ gametes had an average of ~10% per-site CG methylation while MT− gametes had
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an average of ~3%. A large increase in 5meC was observed for all sequence contexts
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during zygote development with 54% (CG) methylation reached by 24 hours.
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Methylation levels began to drop during germination to an average per-site CG
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methylation level of 45%. CHG and CHH methylation levels were lower than CG
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methylation levels in all cases, and this difference was particularly notable in germinated
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zygotes, where CHH and CHG methylation dropped from their peak zygotic levels much
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faster than CG methylation did.
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For all life cycle stages and mating types, methylation of cpDNA is uniformly distributed
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in the chloroplast genome without bias towards genes or other sequence features (Figure
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6B). With the exception of two large inverted repeats in the chloroplast sequence, where
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methylation cannot be measured accurately with short reads, average methylation levels
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tabulated in 1kb intervals rarely deviated more than 5% from the global average,
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suggesting that no specific regions of the chloroplast genome are targeted for
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methylation.
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DNA methyltransferases
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In order to further explore the mechanisms responsible for the dynamic patterns of DNA
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methylation, we identified candidate cytosine methyltransferases from the predicted C.
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reinhardtii proteome based on the presence of predicted DNA cytosine methylase (DCM)
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domains (Materials and Methods). We found a total of six candidate methyltransferases
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with different domain architectures (Supplementary Figure 1) including a homolog of
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Volvox carteri MET1, a putative nuclear methyltransferase (Babinger et al., 2007). As
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described below, three predicted methyltransferases – DMT1a, DMT1b, and DMT4 – had
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expression patterns and/or predicted localization sequences suggesting a role in
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chloroplast DNA methylation. Each of these predicted proteins has DNA methylase as
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well as BAH domains. DMT1a, DMT1b, and DMT4 were all expressed in gametes and
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showed highest levels of expression in zygotes, coinciding with elevated levels of
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methylation in the zygotic samples (Figure 7). DMT1a and DMT1b are near the mating
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locus in its telomere-proximal domain and encode highly similar paralogs. Both genes
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had higher expression in MT+ than in MT− gametes, a pattern which matches the
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methylation bias seen in gametic cpDNA from the two mating types (Figure 6A).
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DMT1a and DMT1b sequences have previously been described as a single gene
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(Nishiyama et al., 2002), but the published cDNA sequence is a hybrid with 5’ sequences
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derived from DMT1a and 3’ sequences from DMT1b (NCBI Gene IDs #5722229 and
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#5722231). Consistent with sub-cellular targeting predictions (Figure 7) the DMT1a pre-
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sequence directs chloroplast localization (Nishiyama et al., 2002). While the MT+ strain
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copy of DMT1a appears to be intact, a survey of structural variants derived from genome
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resequencing data led to the identification of a point insertion in exon 10 of the MT−
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strain copy of DMT1a leading to a frame shift and premature termination before the
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methyltransferase domains (Figure 8B). This insertion was observed in MT–
363
transcriptome data. Furthermore, this point insertion is found in the genome of 12 out of
364
13 MT– strains resequenced by Gallaher et al. No variants predicted to be deleterious
365
were found within the DMT1b gene. Targeting predictions of DMT1b suggest it is
366
mitochondria-localized. Although the mitochondrial genome is largely devoid of
367
cytosine methylation (1-2% global per-cytosine methylation), the zygote sample at 24
368
hours shows evidence of methylation (~13% global per-cytosine methylation)
369
(Supplementary Figure 2). DMT4 is also predicted to encode a chloroplast-targeted
370
cytosine methyltransferase and is one of the 361 genes identified with a strong zygotic
371
expression pattern (Figure 3A), consistent with a possible participation of DMT4 in
372
zygotic cpDNA hypermethylation.
373
374
Discussion
375
376
The dynamic changes in gamete- and zygote-specific mRNA abundance and DNA
377
methylation presented in this work provide a framework for understanding cell
378
differentiation during the Chlamydomonas sexual life cycle. A previous study of plus
379
and minus gamete specific genes focused on cell type genes whose expression was up-
380
regulated during the process of mating (Ning et al., 2013). However, direct comparison
381
with the previous data is confounded by differences in annotation between genome
382
versions used to define transcripts as well as in the different clustering criteria used in the
383
two studies. Here we made use of culture conditions that were designed to suppress the
384
differential transcript abundance signal resulting from growing versus non-growing N-
385
starved cultures to help identify gamete specific transcripts. Our method identified
386
known gametic genes whose expression is mating-type limited (expressed preferentially
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387
in plus versus minus gametes or vice versa). However, because we used nitrogen
388
resupply of stationary phase cultures to create non-growing ‘vegetative’ samples we may
389
have missed highly stable gametic transcripts that did not turn over after nitrogen
390
addition. Nonetheless, nitrogen resupply for several hours was completely effective at
391
suppressing mating, so the transcriptome differences we were able to identify in our
392
gametic versus vegetative samples are likely tied to gametogenesis-related functions and
393
perhaps less relevant for other N-starvation responses such as neutral lipid accumulation.
394
395
Sex-related genes evolve rapidly and are therefore expected to appear younger and have a
396
more restricted phylogenetic distribution than other genes (Swanson and Vacquier, 2002).
397
Indeed, using phylogenomic profiling we found an enrichment of Chlamydomonas-
398
specific genes belonging to the plus or minus gamete up-regulated categories and zygote
399
up-regulated categories (Figure 3C). This finding underscores the importance of species-
400
specific and clade-specific genes as potential drivers of cell-type specialization related to
401
sex and speciation. Although the functions of these genes are difficult to predict since
402
they have no homologs outside of Chlamydomonas or Volvocine algae, we did find
403
enrichment for secretory pathway targeting signals within the plus gamete and zygote
404
predicted proteins (Figure 3B) which could indicate a role for sex-related proteins in the
405
plasma membrane, cell wall or extracellular space. Manual annotation of zygotic genes
406
showed that their predicted functions match processes such as glycosylation and transport
407
that are associated with cell wall formation. In addition we identified or confirmed
408
expression of zygotic genes that may be associated with other poorly-understood
409
differentiation processes, including chloroplast fusion, MT– cpDNA elimination, DNA
410
methylation, and cytoskeletal remodeling (Supplementary Table 1). Deeper investigation
411
of these genes may yield insights into the cell biology of zygote differentiation that may
412
have parallels in other zygospore-forming algae (Brawley and Johnson, 1992) and even
413
in plants where pollen cells must undergo a similar process of dormancy and reactivation
414
when exposed to appropriate conditions (Brown and Lemmon, 2011). Early zygotes in
415
Chlamydomonas also undergo dramatic changes in their cytoskeleton. Unlike the case in
416
animals where paternal but not maternal centrioles are contributed to zygotes (Avidor-
417
Reiss et al., 2015), in Chlamydomonas both parental basal bodies (structurally similar to
15
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418
centrioles) and flagella are retained initially in zygotes, but eventually are disassembled
419
and rebuilt during germination (Cavalier-Smith, 1976). Nonetheless, investigation of
420
conserved zygotically up-regulated cytoskeletal proteins such as LF5 and SF-assemblin
421
may shed light on more general mechanisms involved in controlling the dynamic
422
behavior of flagella/cilia and basal bodies during cellular remodeling and life cycle
423
transitions.
424
425
DNA methylation changes during the life cycle
426
In contrast to many organisms where a large fraction of the nuclear genome is methylated
427
(Smith and Meissner, 2013; Bestor et al., 2014), we found relatively low levels of
428
cytosine methylation in the nuclear genome of Chlamydomonas, consistent with previous
429
surveys that were not as high resolution as reported here (Hattman et al., 1978; Feng et
430
al., 2010). Interestingly, we did identify 23 hypermethylated loci larger than 10kb
431
(Supplementary Table 3) that tended to occur within gene-poor, repeat-rich regions of the
432
genome. However, the mechanism that leads to the methylation of these loci remains
433
unknown. Previous studies on silencing of transgenes in Chlamydomonas found that
434
inserted transgene repeats were frequently methylated, but reported little correlation
435
between methylation and gene expression (Cerutti et al., 1997). On the other hand, in the
436
related colonial alga Volvox carteri where methylcytosine frequency is slightly higher
437
than in C. reinhardtii (1.1% vs. ~0.75%), nuclear methylation did appear to be associated
438
with gene silencing (Babinger et al., 2001; Babinger et al., 2007). Interestingly, V.
439
carteri also has a more repeat-rich genome than C. reinhardtii with many active
440
transposons (Miller et al., 1993; Ueki and Nishii, 2008; Prochnik et al., 2010), and may
441
have retained or evolved higher levels of nuclear methylation activity than C. reinhardtii
442
to suppress transposon activity.
443
444
The low frequency of cytosine methylation in the nuclear genome contrasts with the
445
dynamic and abundant cytosine methylation in the chloroplast genome. The overall
446
patterns we observed are in agreement with previous findings where vegetative cpDNA
447
from both mating types had low levels of cytosine methylation, and that gametes showed
448
elevated levels with MT+ cpDNA having a two or three fold higher frequency of
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449
methylcytosine compared to MT– cpDNA (Royer and Sager, 1979; Dyer, 1982). We
450
observed hypermethylation in zygotes, which has also been seen previously. Our study
451
extended these earlier results by examining genome-wide methylation in cpDNA at
452
single-base resolution. Unlike methylation of nuclear DNA, which was mostly restricted
453
to a few loci, cpDNA cytosine methylation was uniformly distributed across all regions
454
and sequence feature types (genic, intergenic, repeats, exons, introns, etc.). This finding
455
has implications for the function of cpDNA methylation and the enzymes that are
456
responsible for methylating cpDNA (elaborated in next section). Previous models of
457
cpDNA inheritance invoked a methylation-restriction system similar to that in
458
prokaryotes where sequence-specific methyltransferases protect MT+ cpDNA from site-
459
specific restriction endonucleases. However, the non-sequence-specific distribution of
460
methylcytosines we observed and the modest differences between levels of MT+ and
461
MT– cpDNA methylation in gametes are not consistent with a methylation-restriction
462
mechanism. Moreover, if cpDNA methylation were related to methylation and
463
restriction, it would be most effective if it were established at the gametic stage of the life
464
cycle. Instead, the majority of cpDNA methylation occurs in zygotes and is pervasive
465
genome-wide. The purpose of this massive methylation is unknown, but is likely tied to
466
the packaging and protection of cpDNA in zygospores where it may be dormant for many
467
years before germination (Brawley and Johnson, 1992).
468
469
DNA methyltransferases
470
Our survey of candidate methyltransferases revealed two candidates with expression
471
patterns and predicted chloroplast targeting sequences that make them likely responsible
472
for cpDNA methylation—DMT1a and DMT4. Both genes have detectable expression in
473
gametes and zygotes (Figure 7). DMT1a and DMT1b are physically and genetically
474
linked to the mating locus and furthermore, the MT– linked copy of DMT1a has a point
475
mutation that is predicted to generate a truncated, non-functional protein. Although they
476
are linked to the mating locus, the DMT1a/b locus could still recombine with MT or be
477
subject to gene conversion, possibly leading to the creation of strains where levels of
478
gametic cpDNA in MT+ and MT– parents is equivalent. Such strains, if they could be
479
isolated, would be useful for testing the significance of differential gametic cpDNA
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480
methylation and the contribution of the DMT1a gene to gametic cpDNA methylation
481
levels.
482
483
Although the predicted DMT1 and DMT4 encoded methyltransferases have accessory
484
BAH domains that are implicated in protein-protein interactions and targeting to specific
485
loci (Callebaut et al., 1999; Yang and Xu, 2013), the non-specific pattern of cpDNA
486
methylation we observed and attribute to the predicted DMT1a and DMT4 proteins
487
suggest that their targeting is non-sequence-specific. The BAH domains in these proteins
488
may serve other functions such as general targeting of the methyltransferase enzymes to
489
chloroplast nucleoids. The lack of sequence specificity predicted for DMT1a and DMT4
490
may also prove useful for biotechnology applications that require non-specific DNA
491
cytosine methylation activity.
492
493
494
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Materials and Methods
496
Sample generation for bisulfite-treated DNA
497
Chlamydomonas reinhardtii strains R3 (CC-620 R3 NM MT+) and CJU10 (Umen and
498
Goodenough, 2001) were grown in high salt medium (HSM) to concentrations of 3.4 x
499
106 and 3.5 x 106 cells/ml, respectively. 100 ml of each strain culture was used for DNA
500
and RNA isolation as vegetative samples. Cells were collected by centrifugation (3500
501
rpm, 5 min.), resuspended in HSM−N, and after 15 hours, 40 ml of each strain culture
502
was collected as gametic samples. Gametes were mixed and checked for mating
503
efficiency (85% efficient), and after 1 hour, a 40 ml sample was collected, corresponding
504
to the “1 hour zygote” sample. Mixed gametes were split into two flasks: (1) cells in the
505
first were collected after 24 hours, corresponding to the “24 hour zygote sample”; (2)
506
cells in the second were resuspended in water and plated with HSM media, incubated in
507
the light for 24 hours, incubated for 5 days in the dark, and resuspended in Tris-EDTA-
508
NaCl (TEN) buffer with 0.2% NP−40, at which time half of the culture was collected as 6
509
day zygote samples. The remaining half was transferred to TAP medium, incubated in
510
the light for 24 hours, and collected as the germinated zygote sample.
511
512
DNA isolation and purification for bisulfite treatment
513
Cells were resuspended in 4 ml TEN buffer, with the exception of 24 hour and 6 day
514
zygotes. 24 hour and 6 day zygotes were resuspended in 10 ml TEN, mixed for one
515
minute, and repelleted (repeated three times) followed by resuspension in 4 ml TEN. 400
516
μl 20% SDS and 400 μl 20% Sarkosyl were added to each sample. For 24 hour and 6 day
517
zytores, 4 ml zirconium beads was also added followed by vortexing for 5 minutes. 200
518
μl pronase E solution (10 mg/ml) was then added and samples were incubated at 37° C
519
for 30 minutes. 2.5 ml phenol (10 mM Tris-Cl pH 8 saturated) was added, followed by
520
2.5 ml chloroform:isoamylalcohol (24:1). The phases were separated by centrifugation
521
(5000 rpm, 10 min) at 10° C and the upper phase (4.5 ml) was transferred into 9 ml 100%
522
ethanol and incubated overnight at −20° C. Nucleic acids were collected by
523
centrifugation, resuspended in 1 ml 10mM Tris-Cl pH 8 and 100 μl RNase (5 mg/ml) was
524
added. After RNase treatment, samples were extracted again with 1 ml
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525
phenol/chloropform/isoamylalcohol (25:24:1) and DNA was precipitated with 0.3M
526
NaAcetate and 70% isopropanol for 30 minutes at room temperature.
527
528
DNA library preparation
529
500 ng of purified genomic DNA was sheared by sonication with Covaris S2 to generate
530
DNA fragments spanning from 100 to 400 bp size range. Library preparation was carried
531
out using NEBNext DNA Library Prep Master Mix (Set for Illumina, NEB, cat #E6040)
532
according to manufacturer instructions with minor modifications. The ligation was
533
performed using Illumina TruSeq Adapters (Illumina, cat # 15025064), DNA size-
534
selection (200-400 bp range) was carried out with AMPure XP beads (Beckman Coulter,
535
Inc.), prior to bisulfite conversion using EZ DNA Methylation-Lightning Kit (Zymo, cat
536
# D5030). The bisulfite treated DNA was amplified using Illumina Primer Cocktail Mix
537
(Illumina, cat #15027084) and MyTaq Mix (Bioline, cat # BIO-25045) according to the
538
following program: 98°C – 2 min; 12 cycles of: 98°C – 15 sec, 60°C – 30 sec, 72°C – 30
539
sec; 72°C – 5 min.
540
541
RNA library preparation
542
Total RNA isolation for RNA-seq analysis was performed as previously described (De
543
Hoff et al., 2013) with additional DNase treatment (2U Roche RNase-free DNase per 110
544
μg total RNA, 37 °C for 20 min.) before final Qiagen RNeasy column purification.
545
546
Genomic variation
547
Genomic reads were aligned using Burrows-Wheeler Aligner v.0.6.2 v.0.6.2 (Li et al.,
548
2008), with default parameters, to the version 5.0 assembly of the C. reinhardtii CC-503
549
genome (Merchant et al., 2007). After removing duplicates with Picard MarkDuplicates
550
(http://picard.sourceforge.net), we applied Genome Analyzer Tool Kit (McKenna et al.,
551
2010) base quality score recalibration, InDel realignment, and small variant discovery
552
(DePristo et al., 2011). This was followed by hard filtering of variants with extensive
553
manual calibration guided by inspections in IGV (Thorvaldsdottir et al., 2013).
554
555
RNA-sequencing analysis
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556
RNA sequencing data was aligned to the C. reinhardtii genome (assembly version 5.5;
557
Phytozome v10.3 gene annotation) with TopHat v2.0.10 using the annotation to guide
558
spliced alignment. Default parameters were kept, with the exception of constraining
559
intron lengths to less than 5kb. Expression levels were quantified using Cufflinks v2.2.1
560
to compute FPKMs. Sequence data has been deposited in NCBI’s Short Read Archive
561
(SRA) under the accessions SRR2048158, SRR2048159, SRR2048160, SRR2048161,
562
SRR2048162, SRR2048163, SRR2048164, and SRR2048165.
563
564
Identification of gamete- and zygote-specific genes
565
Gamete- and zygote-specific genes were identified by applying a series of filters to the
566
FPKM data generated as described above. We defined gamete-specific genes for each
567
mating type as those that have expression in all samples (excluding the zygote) that is
568
less than 10% of the expression level in the gamete of that mating type. Zygote-specific
569
genes were defined as those whose expression in all other samples was less than 25% of
570
the expression level in the zygote. Expression values for samples with replicates were
571
averaged.
572
573
Bisulfite-treated DNA sequencing analysis
574
Raw sequence data was demultiplexed using standard Illumina barcode indices and was
575
checked for quality using FastQC (v0.10.1). Bisulfite-converted sequences were aligned
576
to the Chlamydomonas reinhardtii nuclear genome (Nov. 2011 assembly) and chloroplast
577
genome using the BS-Seeker2 alignment pipeline v2.0.5 (Guo et al., 2013). Default
578
whole-genome bisulfite alignment parameters were chosen with the following
579
exceptions: Bowtie2 was used as the aligner and local alignments were enabled.
580
Methylation levels were called for cytosines covered by at least 4 reads. Sequence data
581
has been deposited in NCBI’s SRA under the accessions SRR2051057, SRR2051058,
582
SRR2051059, SRR2051060, SRR2051061, SRR2051062, SRR2051063, and
583
SRR2051065.
584
585
Identification of candidate methyltransferases
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586
To identify candidate DNA methyltransferases, protein sequences of C. reinhardtii
587
(Phytozome v10 gene annotation) were scanned against the Pfam-A database (Release
588
27) using the standalone PfamScan scripts provided by the Wellcome Trust Sanger
589
Institute. The presence of the ‘C-5 cytosine-specific DNA methylase’ domain (PF00145)
590
was used as a criterion for assignment as a cytosine methyltransferase.
591
592
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593
Figure Captions
594
595
Figure 1: Chlamydomonas reinhardtii sexual life cycle and sequenced samples
596
597
Vegetative C. reinhardtii cells of each mating type (MT+ and MT−) can be induced to
598
undergo gametogenesis by nitrogen starvation (−N). Gametes of opposite mating type
599
recognize each other through flagellar adhesion and fuse to form a diploid zygote.
600
During zygote maturation MT− chloroplast DNA (cpDNA) is eliminated, flagella are
601
resorbed, and a thick, zygote cell wall forms. Upon return to nitrogen and light
602
zygospores undergo meiosis to form four haploid progeny (two of each mating type, all
603
containing uniparentally inherited MT+ parental cpDNA) that reenter the vegetative
604
cycle. Colored boxes designate samples and material sequenced. Blue and red unfilled
605
circles represent MT+ and MT– chloroplast genomes. Filled pink and light blue circles
606
represent nuclear DNA from MT+ and MT– strains respectively.
607
608
Figure 2: Genetic differences between R3 (MT+) and CJU10 (MT−) parental strains
609
610
(A) Genetic differences, categorized by variant-type (single nucleotide variants [SNVs],
611
insertions, and deletions) are shown with total number of variant loci and total bases. (B)
612
Large-scale view of chromosomes 6 and 17, where 98.5% of the identified variants are
613
located. Locations of sequence differences are shown in red. The region of chromosome
614
6 containing the mating locus is denoted by a blue bar. (C) Schematic representation of
615
the antibiotic resistance transgene insertion in CJU10 chloroplast DNA. The aadA gene,
616
along with the rbcL 5’ promoter and psbC 3’ UTR, is inserted between inverted repeat
617
region “B” (IRB) and the endogenous atpB gene.
618
619
Figure 3: Gamete- and zygote-specific genes
620
621
(A) Box and whisker plots of gene expression profiles for gamete- and zygote-specific
622
genes are plotted. Values are plotted relative to the maximum expression value of each
623
of the genes. 293 and 68 genes were expressed specifically in MT+ and MT– gametes
23
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624
respectively. 627 genes were expressed specifically in zygotes. (B) Localization
625
predictions for proteins encoded by gamete (gam. MT+, gam. MT−) and zygote-specific
626
genes, compared to all predicted proteins in the Chlamydomonas proteome (all). Data are
627
plotted as fraction predicted for each of four compartments with total numbers indicated
628
within each graph portion: secretory in blue; chloroplast (chloro.) in green; mitochondria
629
(mito.) in purple; other in gray. Starred values are significantly different from the total
630
proteome distribution (*: p < 0.05; **: p < 0.01). (C) Predicted protein groups as
631
described in 3B are plotted according to taxonomic distribution of encoded proteins from
632
each category. From bottom (Chlamydomonas-specific proteins, dark green) to top (all
633
cellular organisms, gray) are increasingly broad phylogenetic distributions. Starred
634
samples indicate significant enrichment or depletion in a taxonomic category relative to
635
the distribution of all proteins.
636
637
Figure 4: Functional annotation of proteins encoded by gamete- and zygote-specific
638
genes
639
640
Functional annotations for gamete- and zygote-specific gene lists are shown as bar plots
641
showing the fraction of total proteins in each group with the specified annotation. Total
642
number of genes in each annotation category versus number of genes with the described
643
annotation are shown. Significant differences between all genes and gamete or zygote
644
specific genes were calculated with the hypergeometric test and significant p-values are
645
shown. (A) GreenCut2 genes (Karpowicz et al., 2011; Heinnickel and Grossman, 2013)
646
(B) CiliaCut genes (Merchant et al., 2007). (C) MapMan cell wall related genes (Thimm
647
et al., 2004). (D) MapMan transport related genes.
648
649
Figure 5: Nuclear methylation at different C. reinhardtii life cycle stages
650
651
(A) Bulk averages of 5-methyl cytosine in the nuclear genome are shown for each sample
652
and categorized by the fraction of 5meC in each of three sequence contexts (CG, CHG
653
and CHH). (B) Scaled representations of the 17 C. reinhardtii chromosomes with 23
654
hypermethylated regions larger than 10kb shaded in blue. (C) Plots comparing gene and
24
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655
repeat densities of hypermethylated regions relative to the entire genome. (D) Genome
656
browser display of nuclear cytosine methylation frequency at two representative
657
hypermethylated loci from chromosome 6. Two representative examples of strain-
658
specific hypermethylated loci are shown side-by-side, with a gene track below for
659
genomic context. In the coverage plot, the blue represents unmethylated cytosines
660
(converted by bisulfite treatment), the red represents methylated cytosines (unconverted),
661
and the gray represents the total coverage (including reads from the opposite strand
662
which do not provide methylation information).
663
664
Figure 6: Chloroplast genome methylation at different life cycle stages
665
666
(A) Bulk cytosine methylation frequencies for chloroplast DNA at different life cycle
667
stages are plotted for each methylation context. Mating type of each sample (+, - or
668
diploid +/-) and time of zygote development and germination “(g)” are shown below the
669
graph. (B) Plot of average CG methylation frequency for each sample in 1kb bins across
670
the chloroplast genome. The two inverted repeat regions are shaded in gray. Plots are
671
color coded according to the legend on the right.
672
673
Figure 7: Candidate chloroplast methyltransferases in C. reinhardtii
674
675
The protein domain structure of each candidate methyltransferase is shown schematically
676
(green = DNA methylase domain; red = bromo adjacent homology (BAH) domain).
677
Predicted localizations from PredAlgo and WolfPSORT (Cp=chloroplast,
678
Mt=mitochondria) are shown alongside the log-transformed normalized expression level
679
of each candidate from different RNA-seq samples.
680
681
Figure 8: Single base insertion variant in DMT1a leads to premature stop codon
682
683
(A) The DMT1 locus on chromosome 6 containing DMT1a and DMT1b is shown below
684
its context in the entire chromosome. (B) Upper diagram shows enlarged chromosomal
685
region containing DMT1 locus with genomic coordinates and adjacent genes. The
25
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686
position of the insertion variant in DMT1a is indicated by a yellow triangle. The inset
687
below is an expanded view of exon 10 from DMT1a showing the single-base insertion in
688
the MT– strain (CJU10) and the altered protein coding sequence and premature stop
689
codon caused by the cytosine insertion shown in bold and marked with a yellow triangle.
690
691
Supplementary Figure 1: Candidate methyltransferases in C. reinhardtii
692
693
Candidate methyltransferases were identified by the presence of a DNA
694
methyltransferase domain. Predicted localizations from PredAlgo and WolfPSORT are
695
shown alongside the expression patterns of each candidate methyltransferase, scaled to
696
the maximum and minimum expression values per gene.
697
698
Supplementary Figure 2: Methylation patterns for Chlamydomonas mtDNA
699
700
Cytosine methylation is absent (1-2% per-cytosine methylation) in all samples, with the
701
exception of 24h zygotes (13% per-cytosine methylation). In the coverage plots, blue
702
represents unmethylated cytosines (converted by bisulfite treatment), red represents
703
methylated cytosines (unconverted), and gray represents the total coverage (including
704
reads from the opposite strand which do not provide methylation information).
705
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34
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CJU10 (mt–)
R3 (mt+)
vegetative
cells
Genome (Whole)
Transcriptome (RNA-seq)
1 2
1 2
Methylome (BS-Seq)
gametogenesis
1 2
Replicates
mating
gametic
cells
1 2
fertilization
cell fusion
zygote development
!!
!
!
! !
!
!!
!
!! !
!
new zygote
pooled
(0.5, 1, 2 hrs)
!!
!!
!
!
! !
!
!
!
!
!! !
!
!
!
degradation
of mt– cpDNA
nuclear &
organellar
fusion
1h
2-4 h
!! !
!
!
zygospore
maturation
24 h
meiosis &
cpDNA replication
daughter cells
6d
Figure 1. Chlamydomonas reinhardtii sexual life cycle and sequenced samples
Vegetative C. reinhardtii cells of each mating type (MT+ and MT−) can be induced to undergo gametogenesis by nitrogen starvation
(−N). Gametes of opposite mating type recognize each other through flagellar adhesion and fuse to form a diploid zygote. During
zygote maturation MT− chloroplast DNA (cpDNA) is eliminated, flagella are resorbed, and a thick, zygote cell wall forms. Upon return
to nitrogen and light zygospores undergo meiosis to form four haploid progeny (two of each mating type, all containing uniparentally
inherited MT+ parentalDownloaded
cpDNA) that
reenter the vegetative cycle. Colored boxes designate samples and material sequenced. Blue
from on June 17, 2017 - Published by www.plantphysiol.org
and red unfilled circlesCopyright
represent
MT+
and MT–
chloroplast
genomes.
Filled
pink and light blue circles represent nuclear DNA from
© 2015 American
Society
of Plant Biologists.
All rights
reserved.
MT+ and MT– strains respectively.
A
B
Genetic Differences
R3 (MT+) è CJU (MT–)
Category
mating
locus
Loci
Total Bases
101,452
101,452
Insertions
7,510
35,999
Deletions
8,113
41,209
SNVs
Chr. 6
C
R3 (mt+)
IRB
Chr. 17
atpB
Insertion (cpDNA)
CJU (mt–)
IRB
rbcL
(promoter)
aadA
psbC
atpB
(UTR)
Figure 2. Genetic differences between R3 (MT+) and CJU10 (MT–) parental strains
(A) Genetic differences, categorized by variant-type (single nucleotide variants [SNVs], insertions, and deletions) are shown with total number
of variant loci and total bases. (B) Large-scale view of chromosomes 6 and 17, where 98.5% of the identified variants are located. Locations
of sequence differences are shown in red. The region of chromosome 6 containing the mating locus is denoted by a blue bar. (C) Schematic
representation of the antibiotic resistance transgene insertion in CJU10 chloroplast DNA. The aadA gene, along with the rbcL 5’ promoter and
psbC 3’ UTR, is inserted between inverted repeat region “B” (IRB) and the endogenous atpB gene.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
gamete-specific (mt+)
293 genes
gamete-specific (mt–)
68 genes
ValsOfGenesInNewListsInNewData −− JU ChlReiV5 −− Tophat_to_Cufflinks −− gamPlus
ValsOfGenesInNewListsInNewData −− JU ChlReiV5 −− Tophat_to_Cufflinks −− gamMinus
1.0
0.8
●
gamPlus
gamMinus
zygote
100%
2628
14
78
64
*
**
**
**
**
80%
10
3361
72
45
70%
60%
131
102
19
126
102
10
17
2836
71
55
95
73
9
50%
0.0
vegPlus
vegMinus
gamPlus
C
187
129
*
216
275
**
**
60%
**
40%
50%
50%
gamPlus
7 97
93
**
17
18 17
*
13
15 14
12
6058
6058
50
Secretory
Chloroplast
**
2712
20%
20%
20%
50
68
3
2
11*
*3
8164
81
**
**
**
179
179
**
153**
45
45
46 35
**
46
37
78
**
**
*
78 *
73
34 **
2712 143 **
2712
34
143
191
**
38 **
**
**
198 **
198
131 **
3605
10%
10%
zygote
*
1
5 7
5
2
1300 Mitochondria
1300
1300
878 Others
40%
40%
878
878
30%
30%
gamMinus
**52 **
13
14
70%
70%
60%
60%
vegMinus
21 **
21 **
29
80%
80%
6058
10%
10%
vegPlus
52
80%
50%
Other
20%
43
30
veg. veg. gam. gam. zyg.
(mt+) (mt–) (mt+) (mt–)
3191
90% 90%
90%3191 3191
30%
30%
zygote
100% 100%
100%
70%
40%
8916
gamMinus
veg. veg. gam. gam.
(mt+) (mt–) (mt+) (mt–)
Secretory
vegMinus
●
●
●
●
●
●
●
●
●
●
●
●
0.0
0.0
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
veg. veg. gam. gam.
(mt+) (mt–) (mt+) (mt–)
vegPlus
●
●
●
●
●
Chloro.
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
0.2
0.2
●
●
●
●
●
●
●
●
●
●
●
●
●
0.2
0.4
0.4
0.4
0.6
0.6
0.6
0.4
90%
ValsOfGenesInNewListsInNewData −− JU ChlReiV5 −− Tophat_to_Cufflinks −− zygote
1.0
0.8
0.6
Min
B
zygote-specific
627 genes
0.8
0.8
0.2
Gene Expression
1.0
Max
Mito.
A
**
3605
3605
0%
0%
all
all
+
–
gam. mt minus
gam. mt
plus
plus gamete
gamete minus gamete
gamete
zygote
zygote
zygote
0% all
0%
plus
allall
gam.
mt+
plus
minus
gam.
mt–
minus
zygote
zygote
zygote
Figure 3. Gamete- and zygote-specific genes
(A) Box and whisker plots of gene expression profiles for gamete- and zygote-specific genes are plotted. Values are plotted relative to the
maximum expression value of each of the genes. 293 and 68 genes were expressed specifically in MT+ and MT– gametes respectively. 627
genes were expressed specifically in zygotes. (B) Localization predictions for proteins encoded by gamete (gam. MT+, gam. MT−) and
zygote-specific genes, compared to all predicted proteins in the Chlamydomonas proteome (all). Data are plotted as fraction predicted for
each of four compartments with total numbers indicated within each graph portion: secretory in blue; chloroplast (chloro.) in green;
mitochondria (mito.) in purple; other in gray. Starred values are significantly different from the total proteome distribution (*: p < 0.05; **: p <
0.01). (C) Predicted protein groups as described in 3B are plotted according to taxonomic distribution of encoded proteins from each category.
From bottom (Chlamydomonas-specific proteins, dark green) to top (all cellular organisms, gray) are increasingly broad phylogenetic
distributions. Starred samples indicate significant enrichment or depletion in a taxonomic category relative to the distribution of all proteins.
A
B
GreenCut2 Genes
6%
4%
2.0%
p = 0.0098
p = 0.0018
1.6%
Genes In List
Genes In List
5%
555/
17,741
3%
2%
9/627
1%
1/293
0%
all
C
CiliaCut Genes
gam.
(mt+)
1.2%
1/68
161/
17,741
0.8%
3/627
1/293
0.4%
0/68
gam.
(mt–)
0.0%
zygote
MapMan: "Cell Wall"
all
D
gam.
(mt+)
gam.
(mt–)
zygote
MapMan: "Transport"
2.0%
1.5%
7/559
1.0%
0.5%
p = 1.386 x 10-3
6.0%
Genes In List
Genes In List
p = 7.276 x 10-5
24/559
4.0%
368/
17,302
2.0%
33/
17,302
0.0%
0.0%
all
zygote
all
zygote
Figure 4. Functional annotation of proteins encoded by gamete- and zygote-specific genes
Functional annotations for gamete- and zygote-specific gene lists are shown as bar plots showing the fraction of total
proteins in each group with the specified annotation. Total number of genes in each annotation category versus
number of genes with the described annotation are shown. Significant differences between all genes and gamete or
zygote specific genes were calculated with the hypergeometric test and significant p-values are shown. (A)
GreenCut2 genes (Karpowicz et al., 2011; Heinnickel and Grossman, 2013) (B) CiliaCut genes (Merchant et al.,
2007). (C) MapMan cell wall related genes (Thimm et al., 2004). (D) MapMan transport related genes.
A
B
C
D
veg.(mt+)
veg.(mt–)
gam. (mt+)
gam. (mt–)
zyg. (1h)
zyg. (24h)
zyg. (6d)
zyg. (germ.)
Genes
Figure 5. Nuclear methylation at different C. reinhardtii life cycle stages
(A) Bulk averages of 5-methyl cytosine in the nuclear genome are shown for each sample and categorized by the fraction of 5meC in each of three
sequence contexts (CG, CHG and CHH). (B) Scaled representations of the 17 C. reinhardtii chromosomes with 23 hypermethylated regions larger than
10kb shaded in blue. (C) Plots comparing gene and repeat densities of hypermethylated regions relative to the entire genome. (D) Genome browser
display of nuclear cytosine methylation frequency at two representative hypermethylated loci from chromosome 6. Two representative examples of
strain-specific hypermethylated loci are shown side-by-side, with a gene track below for genomic context. In the coverage plots, blue represents
unmethylated cytosines (converted by bisulfite treatment), red represents methylated cytosines (unconverted), and gray represents the total coverage
(including reads from the opposite strand which do not provide methylation information).
60
CG
CHG
CHH
0
10
% Methylation
20 30 40
50
A
mt
S1
+
–
S3
+
S5
S7
– +/– +/– +/– +/–
1h 24h 6d (g)
time
vegetative gametes
zygotes
B
Figure 6. Chloroplast genome methylation at different life cycle stages
(A) Bulk cytosine methylation frequencies for chloroplast DNA at different life cycle stages are plotted for each methylation
context. Mating type of each sample (+, - or diploid +/-) and time of zygote development and germination “(g)” are shown
below the graph. (B) Plot of average CG methylation frequency for each sample in 1kb bins across the chloroplast genome.
The two inverted repeat regions are shaded in gray. Plots are color coded according to the legend on the right.
Protein
Architecture
Predicted
Localization
Color Key
20
40
60
Life Cycle
Expression
80
100
Value
Cre06.g249350 (dmt1a)
Cp
Cre06.g249500 (dmt1b)
Mt
Cp
80
1
60
2
40
veg.
(mt–)
gam.
(mt+)
gam.
(mt–)
20
V5
veg.
(mt+)
V4
PredAlgo WolfPSORT
V3
DNA methylase domain
Bromo adjacent homology
(BAH) domain
3
Cp
V2
Cp
V1
Cre06.g286650 (dmt4)
0
zyg.
Figure 7. Candidate chloroplast methyltransferases in C. reinhardtii
The protein domain structure of each candidate methyltransferase is shown schematically (green = DNA methylase domain; red = bromo
adjacent homology (BAH) domain). Predicted localizations from PredAlgo and WolfPSORT (Cp=chloroplast, Mt=mitochondria) are shown
alongside the normalized expression level of each candidate from different RNA-seq samples.
FPKM
A
D.
Cre06.g249350.t1.1
dmt1a
Cre06.g249400.t1.1
Cre06.g249500.t1.1
dmt1b
Cre06.g249450.t2.1
B
…………
Exon 10
R3 (mt+)
ATTCCAG GTCCGCCGTTTCCTGCGGCCCGAGGACCTGTGTCCGACCTGGCC
P
CJU (mt–)
P
F
P
A
A
R
G
P
V
S
D
L
A
ATTCCAG GTCCGCCGTTTCCTGCGGCCCGAGGACCTGTGTCCCGACCTGGC
P
P
F
P
A
A
R
G
P
V
S
R
P
G
………
Exon 10
GGCCAGGAGGAGGAGGCTGAGTGGGCG
G
Q
E
E
E
A
E
W
A
CGGCCAGGAGGAGGAGGCTGAGTGGGC
T
R
P
G
G
G
*
Figure 8. Single base insertion variant in DMT1a leads to premature stop codon
(A) The DMT1 locus on chromosome 6 containing DMT1a and DMT1b is shown below its context in the entire chromosome. (B) Upper diagram
shows enlarged chromosomal region containing DMT1 locus with genomic coordinates and adjacent genes. The position of the insertion variant
in DMT1a is indicated by a yellow triangle. The inset below is an expanded view of exon 10 from DMT1a showing the single-base insertion in the
MT– strain (CJU10) and the altered protein coding sequence and premature stop codon caused by the cytosine insertion shown in bold and
marked with a yellow triangle.
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