bioRxiv preprint doi: https://doi.org/10.1101/674895; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 4.0 International license. 1 Phylogenomics provides new insights into gains and 2 losses of selenoproteins among Archaeplastida 3 Hongping Liang1,2,3#, Tong Wei2,3,4#, Yan Xu1,2,3#, Linzhou Li2,3,6, Sunil Kumar Sahu2,3,4, Hongli 4 Wang1,2,3, Haoyuan Li1,2, Xian Fu2,3, Gengyun Zhang2,4, Michael Melkonian7, Xin Liu2,3,4, Sibo 5 Wang2,4,5*,Huan Liu2,4,5* 6 1 BGI Education Center, University of Chinese Academy of Sciences, Beijing, China. 7 2 BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen 518083, China. 8 3 China National Gene Bank, Institute of New Agricultural Resources, BGI-Shenzhen, Jinsha Road, 9 Shenzhen 518120, China. 10 4 State Key Laboratory of Agricultural Genomics, BGI-Shenzhen, Shenzhen 518083, China. 11 5 Department of Biology, University of Copenhagen, Copenhagen, Denmark. 12 6 School of Biology and Biological Engineering, South China University of Technology, 510006, China. 13 7 Botanical Institute, Cologne Biocenter, University of Cologne, Cologne D-50674, Germany. 14 #these authors contributed equally to this work 15 * Correspondence: *[email protected] 16 Abstract: Selenoproteins that contain selenocysteine (Sec) are found in all kingdoms of life. 17 Although they constitute a small proportion of the proteome, selenoproteins play essential roles in 18 many organisms. In photosynthetic eukaryotes, selenoproteins have been found in algae but are 19 missing in land plants (embryophytes). In this study, we explored the evolutionary dynamics of Sec 20 incorporation by conveying a genomic search for the Sec machinery and selenoproteins across 21 Archaeplastida. We identified a complete Sec machinery and variable sizes of selenoproteomes in 22 the main algal lineages. However, the entire Sec machinery was missing in the BV clade 23 (Bangiophyceae-Florideophyceae) of Rhodoplantae (red algae) and only partial machinery was 24 found in three species of Archaeplastida, indicating parallel loss of Sec incorporation in different 25 groups of algae. Further analysis of genome and transcriptome data suggests that all major lineages 26 of streptophyte algae display a complete Sec machinery, although the number of selenoproteins is 27 low in this group, especially in subaerial taxa. We conclude that selenoproteins tend to be lost in 28 Archaeplastida upon adaptation to a subaerial or acidic environment. The high number of redox- 29 active selenoproteins found in some bloom-forming marine microalgae may be related to defense 30 against viral infections. Some of the selenoproteins in these organisms may have been gained by 31 horizontal gene transfer from bacteria. 32 Keywords: Evolution; Horizontal gene transfer; Phylogenomics; Selenoproteins; Selenocysteine; Sec 33 machinery; 34 35 1. Introduction 36 Selenium (Se) is an essential trace element for human health and its deficiency leads to various 37 diseases, such as Keshan and Kashin-Beck diseases, and affects the immune system and promotes 38 cancer development [1,2]. An essential Se metabolism is present in many organisms, including 39 bacteria, archaea, and eukaryotes [1,3,4]. However, higher concentrations of Se are toxic by 40 functioning as a pro-oxidant, which affects the intracellular glutathione (GSH) pool leading to an 41 enhanced level of ROS accumulation [5,6]. Se is essential for growth and development of numerous 42 algal species but not for terrestrial plants (embryophytes), although it accumulates in certain plant 43 species and can serve as dietary sources for Se uptake [3,7-9]. 44 Se is incorporated into nascent polypeptides in the form of selenocysteine (Sec), the 21st amino 45 acid [10]. Se incorporation requires a specialized machinery and Sec insertion sequence (SECIS) 46 elements present in selenoprotein mRNAs [11,12]. In eukaryotes, it consists of Sec synthesis and Sec 47 incorporation. Sec synthesis starts with tRNASec, aminoacylated with serine, which is phosphorylated bioRxiv preprint doi: https://doi.org/10.1101/674895; this version posted July 1, 2019. 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 4.0 International license. 48 by O-phosphoseryl-transfer tRNASec kinase (PSTK) and then catalyzed by Sec synthase (SecS) to 49 produce selenocysteinyl-tRNASec from selenophosphate [10-13]. The Sec donor, selenophosphate, is 50 generated from selenide by selenophosphate synthetase 2 (SPS2), which is often a selenoprotein itself 51 [14,15]. During Sec incorporation, SECIS-binding protein 2 (SBP2) recognizes the SECIS elements in 52 the 3’-untranslated region (3’-UTR) and recruits the Sec-specific elongation factor (eEFSec) that 53 delivers selenocysteinyl-tRNASec to the ribosome at the in-frame Sec-coding UGA stop codon (Figure 54 2a). Bacteria possess a similar machinery including selB (Sec-specific elongation factor), selC (tRNASec) 55 and selD (selenophosphate synthase), except that Sec synthesis is catalyzed by a single bacterial Sec 56 synthase, SelA [10]. 57 Although selenoproteins constitute only a small fraction of the proteome in any living organism, 58 they play important roles in redox regulation, antioxidation, and thyroid hormone activation in 59 animals including humans [16]. Sec incorporation has been well documented in animals, bacteria, 60 and archaea, while the largest selenoproteome was reported in algae. In the pelagophyte alga 61 Aureococcus anophagefferens, 59 selenoproteins were identified in its genome, compared with 25 62 selenoproteins in humans [17,18]. The green alga Chlamydomonas reinhardtii has at least ten 63 selenoproteins, whereas the picoplanktonic, marine green alga Ostreococcus lucimarinus harbors 20 64 selenoprotein genes in its genome [3,19]. Considering that Se is essential for growth in at least 33 algal 65 species that belong to six phyla, Sec incorporation is thought to be universal in diverse algal lineages 66 [20]. In a previous study, no selenoproteins were found in any land plants [7], suggesting a complete 67 loss of Sec incorporation after streptophyte terrestrialization. Exploring the Sec machinery across the 68 Archaeplastida, especially in algae, would provide insight into its evolutionary dynamics in this 69 important lineage of photosynthetic eukaryotes. Here in this study, we searched 38 plant genomes, 70 including 33 algal species that represent the major algal lineages, for the Sec machinery and 71 selenoproteins. 72 2. Results 73 bioRxiv preprint doi: https://doi.org/10.1101/674895; this version posted July 1, 2019. 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 4.0 International license. 74 2.1. Sec machinery in algae 75 To cover the plant tree of life, we selected 33 genomes of algal species and 5 embryophyte species 76 with a focus on Archaeplastida, the major group of photosynthetic eukaryotes with primary plastids. 77 The 33 algal species include one glaucophyte, six rhodophytes, 16 chlorophytes, and seven 78 streptophyte algae. Another three species, the pelagophyte A. anophagefferens, the diatom Thalassiosira 79 pseudonana, and the coccolithophorid Emiliania huxleyi, were also included to represent other distinct 80 algal lineages (Supplementary Table S1A). 81 The Sec machinery was searched in 38 genome assemblies using Selenoprofiles [21] (See 82 Methods). As shown in Figure 1, embryophytes lack the entire Sec machinery as previously reported 83 [7]. Interestingly, the Sec machinery is not intact in all tested algal species. Among 33 algae, three 84 Rhodoplantae lack the entire Sec machinery as in embryophytes. The chlorophyte Monoraphidium 85 neglectum, and the rhodophyte Cyanidioschyzon merolae lack PSTK, and the glaucophyte Cyanophora 86 paradoxa SBP2. According to the species tree, it seems that the Sec machinery was lost completely in 87 one rhodophyte clade that includes Porphyra umbilicalis, Pyropia yezoensis, and Chondrus crispus and 88 partially in a few other algal species (Figure 1). 89 90 91 Figure 1. The number and distribution of selenoproteins, and enzymes involved in the Sec 92 machinery. The phylogenetic tree was retrieved from the NCBI taxonomy database and the 1 KP 93 Project (http://www.onekp.com). Presence (green symbols) or absence (empty symbols) of the 94 enzymes involved in the Sec machinery (circles) and tRNASec (triangles) across sequenced 95 embryophyte, streptophyte algae, chlorophyte, Rhodoplantae, Glaucoplantae and protist 96 genomes are shown in the left panel. The distribution and number of selenoproteins are plotted 97 in the yellow column in the second panel, and the predicted SECIS elements are represented by 98 the blue bars. Distribution and number of selenoprotein homologues (Cys) are plotted in an 99 orange column on the right panel. Prasinophyte algae (Mamiellophyceae) are highlighted in red. 100 2.2. Sec incorporation in the major algal lineages 101 In addition, we also identified the complete Sec machinery in some Rhodoplantae (Figure 1). 102 The Rhodoplantae are often classified at the subphylum level into two clades,