bioRxiv preprint doi: https://doi.org/10.1101/2021.08.13.456063; this version posted August 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Gametogenesis in the green seaweed Ulva mutabilis coincides with massive 2 transcriptional restructuring 3 4 Xiaojie Liu1, Jonas Blomme1,2,3, Kenny Bogaert1, Sofie D’hondt1, Thomas Wichard4, 5 Olivier De Clerck1∗ 6 7 Affiliations: 8 1 Phycology Research Group and Center for Molecular Phylogenetics and Evolution, 9 Ghent University, Gent, Belgium 10 2 Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 11 Ghent, Belgium 12 3 VIB Center for Plant Systems Biology, 9052 Ghent, Belgium 13 4 Institute for Inorganic and Analytical Chemistry, Jena School for Microbial 14 Communication, Friedrich Schiller University Jena, Jena, Germany 15 16 Email addresses: 17 Xiaojie Liu: [email protected] 18 Jonas Blomme: [email protected] 19 Kenny Bogaert: [email protected] 20 Sofie D’hondt: [email protected] 21 Thomas Wichard: [email protected] 22 Olivier De Clerck: [email protected] (Corresponding author) 23 24 Date of submission: 2021-08-12 25 Number of main figures: 6 26 Number of main tables: 1 27 Word count: 3486 28 Number of figures in supplementary files: 2 29 Number of tables in supplementary files: 6 30 31 Running title: Ulva gametogenesis transcriptional framework 32 33 Highlight: Transcriptomic analyses of gametogenesis in the green seaweed Ulva 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.13.456063; this version posted August 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 34 highlight the importance of a conserved RWP-RK transcription factor in induction of 35 sexual reproduction. 36 37 38 Abstract 39 The molecular mechanism underlying sexual reproduction in land plants is well 40 understood in model plants and is a target for crop improvement. However, unlike 41 land plants, the genetic basis involved in triggering reproduction and gamete 42 formation remains elusive in most seaweeds, which are increasingly viewed as an 43 alternative source of functional food and feedstock for energy applications. Here, 44 gametogenesis of Ulva mutabilis, a model organism for green seaweeds, is studied. 45 We analyze transcriptome dynamics at different time points during gametogenesis 46 following induction of reproduction by fragmentation and removal of sporulation 47 inhibitors. Analyses demonstrate that 45% of the genes in the genome are 48 differentially expressed during gametogenesis. We identified several transcription 49 factors that potentially play a key role in the early gametogenesis of Ulva given the 50 function of their homologs in higher plants and microalgae. In particular, the detailed 51 expression pattern of an evolutionary conserved transcription factor containing an 52 RWP-RK domain suggests a key role during Ulva gametogenesis. The identification 53 of putative master regulators of gametogenesis provides a starting point for further 54 functional characterization. 55 56 57 Keywords: Ulva, green seaweeds, reproduction, gametogenesis, transcriptome, 58 RWP-RK transcription factor 59 Introduction 60 Sexual reproduction is one of the most important and conserved processes in 61 eukaryotes. In essence, sexual reproduction encompasses the fusion of two haploid 62 gametes of opposite sex to form a diploid zygote. Zygote formation is either followed 63 by meiosis to restore the haploid state (zygotic meiosis) or by development of a 64 diploid life stage through mitotic divisions (gametic meiosis) (Coelho et al., 2007). 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.13.456063; this version posted August 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 65 Molecular genetic studies of reproduction in land plants resulted in an excellent 66 understanding of the fundamental biological principles and genes involved. However, 67 unlike land plants, the molecular mechanisms involved in the onset of reproduction, 68 the formation of gametes (gametogenesis), fusion and meiosis remain elusive for 69 most algae and seaweeds. In the green algal lineage, the molecular mechanisms 70 underlying gametogenesis and fusion have been extensively studied in 71 Chlamydomonas reinhardtii, a unicellular freshwater green alga (Abe et al., 2004; 72 Abe et al., 2005; Goodenough et al., 2007; Lin and Goodenough, 2007), but to which 73 extent key regulatory genes are conserved across green algae or even land plants 74 remains unclear. 75 Here, we study the expression of genes during the various stages of gamete 76 formation in Ulva, an emerging model for green seaweeds (Balar and Mantri, 2020; 77 De Clerck et al., 2018; Wichard et al., 2015). Ulva species are abundant in coastal 78 benthic communities around the world and form a potential source of biomass that 79 can be used for food, feed, biofuel and nutraceuticals (Alsufyani et al., 2014; Balar 80 and Mantri, 2020; Bolton et al., 2016; Neori et al., 2004; Nisizawa et al., 1987; Wang 81 et al., 2019). Given its commercial value, reproducibly controlling the life cycle of Ulva 82 is important in the development of this crop. Under natural conditions, large parts of 83 the Ulva thallus are converted into gametangia or sporangia in mature individuals 84 and the entire cell content is converted into reproductive cells. Numerous studies 85 have investigated the effect of different endogenous (e.g. production of sporulation 86 and swarming inhibitors) and environmental factors (e.g. light, temperature or nutrient 87 conditions) on gametogenesis in different Ulva species (Luning et al., 2008; Mantri et 88 al., 2011; Sousa et al., 2007; Stratmann et al., 1996; Wichard and Oertel, 2010). 89 Although we are still far removed from a complete understanding of the interplay of 90 the various factors that appear to play a role, thallus fragmentation has been 91 recognized as the most efficient way to induce gametogenesis (Gao et al., 2010; 92 Hiraoka and Enomoto, 1998; Stratmann et al., 1996; Vesty et al., 2015; Zhang et al., 93 2011). 94 The ability to efficiently induce gametogenesis in laboratory cultures of Ulva 95 mutabilis provides an elegant time course to study both phenotypic and molecular 96 factors involved in this process (Wichard and Oertel, 2010). Two sporulation inhibitors, 97 SI-1 and SI-2 control the onset of gametogenesis of Ulva mutabilis. SI-1 is a species- 98 specific glycoprotein secreted in the culture medium which depletes with maturation 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.13.456063; this version posted August 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 99 of the thallus. SI-2 is a nonprotein molecule existing within the inner space between 100 the two blade cell layers. Thallus fragmentation followed by a washing step whereby 101 spent culture medium is replaced, results in the total removal of sporulation inhibitors 102 and induces gametogenesis (Stratmann et al., 1996). Following fragmentation and 103 washing, SI-1 and SI-2 are removed and vegetative cells enter a determination phase 104 (~0-26h, Fig. 1) in which a 'swarming inhibitor' (SWI) is produced (Wichard and 105 Oertel, 2010). SWI functions as a mechanism to synchronize gamete release. The 106 start of the differentiation phase coincides with the completion of the S-phase and the 107 G2-phase (~36 h) in the first cell cycle (Fig. 1). At the end of the determination phase, 108 the cells enter the next S-phase and become irreversibly committed to gametangium 109 differentiation. The differentiation phase (~26-70h) is characterized by a reorientation 110 of the chloroplast, followed by four consecutive cell divisions forming sixteen 111 progametes per cell and their maturation. Mature gametes are eventually released 112 during the swarming phase, following a light stimulus or if the SWI declines in 113 concentration on the third day after induction (Kessler et al., 2017; Wichard and 114 Oertel, 2010). Given that the whole process of Ulva gametogenesis, can be 115 subdivided in discrete phases, the gene regulatory networks involved in the different 116 stages of gametogenesis should be distinct. As a first step toward understanding the 117 molecular mechanisms of gametogenesis, we established the transcriptional 118 structure for Ulva mutabilis gametogenesis in this research. We make use of 119 differential removal of SI’s to separate the effect of fragmentation on transcription 120 from gametogenesis. Transcription patterns are compared with those of other green 121 algae and land plants. 122 Materials and methods 123 Strains and Culture Conditions 124 Haploid gametophytes of Ulva mutabilis [strain 'wildtype' (wt-)] were collected 125 initially in southern Portugal (Føyn, 1958). Gametophytes of U. mutabilis were raised 126 parthenogenetically from unmated gametes and cultured in transparent plastic boxes 127 containing 2 L Provasoli enriched seawater (PES) medium (Provasoli, 1968) with the 128 following specific parameters: light intensity, 70 µmol photons m-2·s-1; temperature, 18 129 ± 1 °C; and 17:7 h light:dark cycle. The medium was completely changed every 2 130 weeks until fertility (~10 weeks). Afterwards, the medium was partially (20 %) 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.13.456063; this version posted August 13, 2021.