{"paper_id":"1abbd2c4-3b72-4f41-817f-b28e72517546","body_text":"1 \n \nChlorophyllide a oxygenase (CAO) gene duplication across the 1 \nViridiplantae 2 \n 3 \n 4 \nMackenzie C. Poirier1, Roberta Wright1, Marina Cvetkovska1*  5 \n1Department of Biology, University of Ottawa, Ottawa, ON, Canada 6 \n 7 \n*Corresponding author:  8 \nMarina Cvetkovska  9 \nDepartment of Biology, University of Ottawa, 30 Marie-Curie Pr., Ottawa, ON, Canada, K1N 10 \n6N5 11 \nEmail: mcvetkov@uottawa.ca  12 \nORCID: 0000-0002-7080-8203 13 \n 14 \n 15 \n 16 \n 17 \n 18 \n 19 \nStatements and Declarations 20 \nCompeting Interests: The authors report that there are no competing interests to declare. 21 \nAcknowledgements: This project was supported by Natural Sciences and Engineering Research 22 \nCouncil of Canada Discovery Grants (NSERC DG) awarded to M.C. The authors are grateful for the 23 \nsupport from the Canada Foundation for Innovation (CFI) and University of Ottawa start-up funding. 24 \nM.P. was supported by Ontario Graduate Scholarship (OGS), NSERC Graduate Scholarship, and 25 \nPolar Knowledge Canada Antarctic Doctoral Scholarship. 26 \nAuthor’s contributions: M. Poirier and M. Cvetkovska conceptualized the work and designed the 27 \nexperiments. Material preparation, data collection and analysis were performed by M. Poirier, R. 28 \nWright and M. Cvetkovska. The first draft of the manuscript was written by M. Poirier and all authors 29 \ncommented on all versions of the manuscript. All authors read and approved the final manuscript. 30 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n2 \n \nAbstract  31 \nViridiplantae, a diverse group of green plants and alga that have evolved from a common ancestor, 32 \nare unified in their ability to produce and use two types of chlorophyll (chlorophyll a and chlorophyll 33 \nb) to capture light energy. In addition to playing a role in light harvesting, chlorophyll b is required at 34 \nthe appropriate level for the accumulation, assembly, and stability of light harvesting complexes 35 \nwithin the photosynthetic apparatus. Chlorophyll b is synthesized from chlorophyll a by the enzyme 36 \nchlorophyllide a oxygenase (CAO), a Rieske-type mononuclear non-heme iron oxygenase.  A 37 \nregulatory degron sequence, described in detail only in land plants, regulates the stability of CAO 38 \nproteins based on the availability of chlorophyll b. Recent identification of CAO gene duplication in 39 \nbryophyte and green algal species, combined with expanded availability of sequenced genomes 40 \nwithin the Viridiplantae, prompted further investigation into the role of gene duplication in the 41 \nevolution of chlorophyll b biosynthesis. Examination of genomes from 246 plant and algae species 42 \nrevealed independently occurring CAO duplications throughout the Viridiplantae, with a higher 43 \nprevalence of duplication in land plants compared to their algal relatives. Additionally, we 44 \ndemonstrate that the degron sequence is poorly conserved in chlorophytes, but first appears as a 45 \nconserved sequence in charophytes, and is very highly conserved among the embryophytes. The 46 \nevolutionary history and functional role of CAO throughout the Viridiplantae lineage is discussed 47 \nbased on these key observations, adding to our understanding of chlorophyll b biosynthesis and 48 \nthe role of CAO in photosynthetic species.  49 \n 50 \nKeywords 51 \nChlorophyll biosynthesis, chlorophyllide a oxygenase, gene duplication, degron, plants and algae 52 \n 53 \n 54 \n 55 \n 56 \n 57 \n 58 \n 59 \n 60 \n 61 \n 62 \n 63 \n 64 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n3 \n \nIntroduction 65 \nPhotosynthetic organisms form the base of most food chains and provide the organic carbon 66 \ncompounds that support life in our biosphere. The capture of light energy by chlorophyll is the first 67 \nstep in the process of photosynthesis that fixes atmospheric CO2 into stable organic compounds. 68 \nOverwhelming evidence indicates that oxygenic eukaryotic photosynthesis that occurs in the 69 \nchloroplasts of modern-day plants and algae traces its origins from an endosymbiotic event with 70 \ncyanobacteria-like prokaryotes (Blankenship 2010; Cardona 2019; Sánchez-Baracaldo and 71 \nCardona 2020). Viridiplantae (or green plants) have primary chloroplasts derived from an ancient 72 \nendosymbiosis, and  encompass two major clades: the chlorophytes and the streptophytes. 73 \nChlorophytes are a monophyletic group of marine, freshwater, and terrestrial green algae. With  74 \n~8,000 described species (Guiry 2024), this group encompasses a large diversity of adaptations, 75 \nmorphologies, and life histories (Leliaert et al. 2012). Streptophyta includes the charophytes, 76 \n~5,500 species of largely freshwater green algae (Guiry 2024) and the embryophytes, or land 77 \nplants. Land plants are thought to have diverged from a single clade within the streptophytes (de 78 \nVries and Archibald 2018) and number ~430,000 species (Ruggiero et al. 2015). One characteristic 79 \nthat unifies members of the Viridiplantae is their ability to synthesize and use two types of 80 \nchlorophylls: chlorophyll a (Chl a) and chlorophyll b (Chl b).  81 \nThe photosynthetic apparatus in the Viridiplatae is derived from the ancestral cyanobacterial 82 \nendosymbiont, which used chlorophyll as the main pigment in their photosystems (Xiong and 83 \nBauer 2002; Sánchez-Baracaldo and Cardona 2020). Chl a is ubiquitously present in all oxygenic 84 \nphotosynthetic eukaryotes, and functions in both energy capture in the antenna light harvesting 85 \ncomplexes (LHCs) and in driving electron transfer in the photosystem II (PSII) and photosystem I 86 \n(PSI) reaction centers. Chl b is specific to the Viridiplantae although it has also been detected in 87 \nprochlorophytes and Acaryochloris, unique photosynthetic prokaryotic groups that lack typical 88 \ncyanobacterial phycobilin light-harvesting pigments (Palenik and Haselkorn 1992; Roche et al. 89 \n1996; Tomitani et al. 1999; Partensky et al. 2018). Chl b resides predominantly in the peripheral 90 \nLHCs (Neilson and Durnford 2010) although it has been detected in the core complexes of some 91 \ndeep water marine chlorophytes  (Kunugi et al. 2016).  92 \nStrict Chl a/b stoichiometry is required for optimal energy transfer during photosynthesis. 93 \nMaintaining the correct Chl a/b ratios is a dynamic process and indicative of adaptation to different 94 \nlight conditions  (Tanaka and Tanaka 2007). In addition to playing a role in light harvesting, Chl b is 95 \nrequired at appropriate levels for the accumulation, assembly, and stability of LHCs in algae 96 \n(Bujaldon et al. 2017), bryophytes (Zhang et al. 2023), and angiosperms (Król et al. 1995; Reinbothe 97 \net al. 2006; Kim et al. 2009; Nick et al. 2013). Under low light conditions, the amount of Chl b 98 \nrelative to Chl a increases, leading to larger LHC antenna size, which maximizes the surface area 99 \nfor light absorption  (Kunugi et al. 2016; Kume et al. 2018; Ueno et al. 2019). Thus, the amount of 100 \nChl b is a major regulator of light-harvesting capacity.  101 \nChlorophyll biosynthesis and turnover occurs through a complex multistep pathway (reviewed in 102 \nBrzezowski et al. 2015; Willows 2020). Chl b is synthesized from Chl a, via the intermediate 7-103 \nhydromethyl chlorophyll a, by the action of a single enzyme: chlorophyllide a oxygenase (CAO) 104 \n(Tanaka et al. 1998; Espineda et al. 1999; Oster et al. 2000; Mueller et al. 2012). Chl b can be 105 \nconverted back into Chl a by chlorophyll b reductase (CBR) and 7-hydroxymethyl reductase (HCAR) 106 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n4 \n \nto complete the cycle (Figure 1; reviewed in detail in Tanaka and Tanaka, 2019). The chlorophyll 107 \ncycle plays an essential role in many biological processes, including biogenesis of LHCs, antenna 108 \nsize regulation during light acclimation, and chlorophyll degradation during senescence. While 109 \nmany of the intricacies of how this pathway is regulated are still not fully understood (Tanaka and 110 \nTanaka 2019), it has been suggested that CAO levels and activity are key determinants of Chl b 111 \naccumulation.  112 \nFigure 1. The chlorophyll cycle in land plants. Chlorophyll a is converted to chlorophyll b via the 113 \nintermediate 7-hydromethyl chlorophyll a, by the action of the enzyme chlorophyllide a oxygenase 114 \n(CAO). In a negative feedback loop, high levels of chlorophyll b trigger the binding of a stromal Clp 115 \nprotease to the degron motif (QDLLTIMILH) and the subsequent destabilization of CAO (dashed red 116 \nline). Chlorophyll b is necessary for the stabilization of Light Harvesting Complexes (LHC) in plants 117 \n(dashed blue line). Excessive amounts of LHC or accumulation of energetically uncoupled non-118 \nfunctional LHCs (e.g., during senescence), triggers the accumulation of Chlorophyll b reductase 119 \n(CBR; blue line) and the conversion of chlorophyll b to chlorophyll a via the action of CBR and 7-120 \nhydroxymethyl reductase (HCAR). This regulatory mechanism of CAO and CBR enables the fine- 121 \ntuning and optimization of chlorophyll b and LHC levels (adapted from Tanaka and Tanaka 2019). 122 \nCAO is part of the Rieske-type mononuclear non-heme iron oxygenase group of proteins (Gray et al. 123 \n2004). It catalyzes the conversion of the methyl group of Chl a into a formyl group to form Chl b 124 \nthrough two oxygenation reactions, likely using ferredoxin as a reductant (Oster et al. 2000). CAO 125 \nhas three main regions: an N-terminal regulatory domain and a C-terminal catalytic domain, which 126 \nare connected by a more variable linker region (Nagata et al. 2004). The catalytic domain contains a 127 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n5 \n \nRieske cluster and a mononuclear iron-binding domain, both of which are necessary for the 128 \nenzymatic activity of CAO (Yamasato et al. 2005; Kunugi et al. 2013). The regulatory domain has 129 \nbeen characterized in detail only in land plants. In Arabidopsis, a conserved sequence 130 \n(QDLLTIMILH) termed a degron was demonstrated to regulate Chl b accumulation by affecting CAO 131 \nprotein turnover. In a negative feedback loop, high levels of Chl b trigger the binding of a stromal 132 \nClp protease to the degron motif leading to destabilization of the CAO protein; this, in turn, halts 133 \nChl b biosynthesis (Sakuraba et al., 2009; Nakagawara et al., 2007). 134 \nThe  evolutionary origins of CAO can be traced back to prochlorophytes, which encode a CAO gene 135 \nhomolog (Tomitani et al. 1999; Nagata et al. 2004). Notably, the prokaryotic CAO-like enzymes lack 136 \nthe regulatory N-terminal domain, suggesting that the Chl b-dependent mechanism for controlling 137 \nCAO stability was acquired in eukaryotes. Recent work on 9 chlorophyte, 2 charophyte, and 3 138 \nembryophyte species suggested that the degron recognition sequence first appeared in 139 \ncharophytes as an adaptation to high-light environments in shallow water (Kunugi et al. 2016). 140 \nThese conclusions, however, were based on a very limited sample of representative species and 141 \nthe presence of a degron in the CAO sequence in charophytes was inferred based on a single 142 \nspecies (Klebsormidinium flaccidum). This hypothesis requires further validation.  143 \nThe CAO protein is encoded by a single gene in most plants and algae (Kunugi et al. 2016; 144 \nSchumacher et al. 2022), with only a very few reported exceptions. Two CAO genes were detected 145 \nin rice (Lee et al. 2005) although only one of them is necessary for Chl b synthesis (Jung et al. 2021). 146 \nRecent work revealed CAO duplication in the bryophyte Physcomitrium patens, where both genes 147 \ncontribute to Chl b accumulation (Zhang et al. 2023). CAO duplication was also reported in two 148 \nAntarctic Chlamydomonas species , where the CAO paralogs in each species appear to be derived 149 \nfrom independent duplications events rather than an ancestral one (Cvetkovska et al. 2019; Poirier 150 \net al. 2025).   151 \nThese insights prompted us to further examine the CAO gene content and the presence of the 152 \nregulatory domain across the diverse plant and algal groups. A systematic examination for the 153 \npresence of CAO across the Viridiplantae has not yet been preformed. In this work we took 154 \nadvantage of the recent increase in publicly available plant and algal genomes to examine the 155 \noccurrence of CAO genes in diverse species across Viridiplantae and demonstrate a widespread 156 \noccurrence of CAO duplicates in this group. Furthermore, building on the work by Kunugi et al. 157 \n(2016), we have examined the occurrence of a degron sequence in 326 unique CAO sequences in 158 \nalgae and plants. We demonstrate that this regulatory region is poorly conserved in chlorophytes, 159 \ncompared to charophyte and embryophyte CAO sequences, suggesting a different mechanism for 160 \nChl b regulation in early aquatic lineages.  161 \nMaterials and Methods 162 \nCAO sequence identification and analyses 163 \nTo identify CAO sequences and duplication events with high accuracy, we used publicly available, 164 \nhigh quality, and fully annotated genomes. Chlorophyte and streptophyte genomes were retrieved 165 \nfrom Phycocosm (Grigoriev et al. 2021), while embryophyte genomes were obtained from 166 \nPhytozome v13 (Goodstein et al. 2012). Putative CAO genes were identified with a tBLASTn search 167 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n6 \n \nwith the Chlamydomonas reinhardtii (CrCAO; Cre01.g043350) and the Arabidopsis thaliana 168 \n(AtCAO; AT1G44446) full-length CAO peptides (Merchant et al. 2007; Lamesch et al. 2012). A strict 169 \ne-value cutoff of ≤e-30 was taken as a threshold, to avoid many unspecific hits due to the 170 \nconserved nature of the Fe-binding and Rieske cluster domains present among diverse plant and 171 \nalgal proteins (Schmidt and Shaw 2001; Ferraro et al. 2005; Przybyla-Toscano et al. 2021). Putative 172 \nCAO sequences were manually curated and truncated or low-quality sequences were excluded 173 \nfrom downstream analyses. Curated CAO sequences were aligned using MUSCLE (Edgar 2004). 174 \nThe degron sequence (QDLLTUMILH) was identified by multiple sequence alignment of the full-175 \nlength CAO amino acid sequences and the regulatory N-terminal domain. In accordance with 176 \nSakuraba et al. 2009, we identify the degron as “highly conserved” if 8 out of the 10 amino acids are 177 \nperfectly conserved, “moderately conserved” if 4 to 8 amino acids are conserved, and “poorly 178 \nconserved” if less than 4 amino acids are conserved. Sequence logos for each major group 179 \n(chlorophyte algae, charophyte algae, non-vascular plants and vascular plants) were generated by 180 \nGeneious Prime 2024.11 (Dotmatics), where the size of the letter reflects its frequency. To examine 181 \nthe expression of CAO duplicate genes, we screened previously published transcriptomes from 16 182 \nspecies with representatives from the Chlorophytes (Arriola et al. 2018), Charophytes (Cheng et al. 183 \n2019), non-vascular (Perroud et al. 2018; Healey et al. 2023) and vascular plants (Zhu et al. 2019; 184 \nWang et al. 2021; Liu et al. 2022b; Shang et al. 2023; Guo et al. 2025; He et al. 2025; Mascuñano et 185 \nal. 2025; Roy et al. 2025). Only transcriptomes that report FPKM, RPKM and TPM values obtained 186 \nfrom organisms cultivated at optimal conditions were considered (Supplementary Table S4).  187 \nPhylogenetic Inference 188 \nThe analyses described above resulted in 374 individual CAO sequences (Supplementary Table 189 \nS1). To avoid inaccurate inference and over-representation, we manually removed heterodimeric 190 \nCAO proteins, where the Rieske domain and the Fe-binding domain are located on different genes 191 \n(Kunugi et al. 2013) (Supplementary Table S1). We also removed identical CAO sequences isolated 192 \nfrom algal cultures identified as the same species but belonging to different strains or culture 193 \ncollections (Scenedesmus obliquus, Auxenochlorella protothecoides, Chlorella sorokiniana, 194 \nOstreococcus tauri, Mesostigma viride, Zygnema circumcarinatum). This resulted in a 280 amino 195 \nacid alignment of 326 unique CAO sequences across 246 species.  196 \nThese manually curated CAO genes were translated into amino acid sequences, aligned with 197 \nMUSCLE and trimmed to remove gaps and ambiguously aligned regions with Gblock (Castresana 198 \n2000; Talavera and Castresana 2007). Maximum likelihood trees were inferred in the CIPRES 199 \nScience Gateway (Miller et al. 2015) using RAxML v8.0 (Stamatakis 2014) with 1000 bootstraps with 200 \nthe Whelan Goldman matrix for globular proteins (WAG), a gamma shape parameter, and empirical 201 \nestimation of invariable sites.  The trees were rooted using prokaryotic CAO from Acaryochloris 202 \nthomasi (WP_110987895.1) and Prochlorotrix hollandica (WP_017713323.1) as an outgroup. 203 \nTandem duplications were defined as those located on the same chromosome or contig within <10 204 \ngenes of each other, based on the genomic coordinates retrieved from Phytozome v13 or 205 \nPhycocosm. To identify ancestral and lineage-specific duplications, we used Notung v2.9 (Stolzer 206 \net al. 2012) and reconciled the CAO trees with a reference species tree derived from NCBI 207 \ntaxonomy (Schoch et al. 2020) via the PhyloT v2 tool (https://phylot.biobyte.de). Putative ancestral 208 \nduplications were only reported when occurring on branches with bootstrap support ≥85%. All 209 \ntrees were visualized  in iTOL v6.8.2 (Letunic and Bork 2021). Previously inferred taxonomic 210 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n7 \n \ninformation for chlorophytes and streptophytes was confirmed via AlgaeBase, University of Galway 211 \n(www.algaebase.org), and for land plants via the Plants Of the World Online, Royal Botanic 212 \nGardens, Kew (https://powo.science.kew.org).  213 \nResults and Discussion 214 \nOccurrences of multiple CAO genes are widespread among the Viridiplantae 215 \nOur analysis demonstrates a widespread occurrence of CAO gene duplication across the 216 \nViridiplantae. Multiple gene copies are more prevalent among the angiosperms, where ~41% of 217 \nexamined species encode for more than one CAO gene. In comparison, we detected two CAO gene 218 \ncopies in only ~11% of chlorophyte and ~20% of charophyte species (Table 1; Table 2, 219 \nSupplementary Table S1; Supplementary Table S3). All 23 species where we detect more than two 220 \nCAO genes are members of the Embryophyta. The modern-day cultivar of sugarcane (Saccharum 221 \nsp. R570) encodes for 6 CAO copies, the largest number we report. This species is a result of inter-222 \nspecific hybridization (Saccharum officinarum x spontaneum; Dumont et al. 2022), which has 223 \nresulted in a very large and highly redundant genome (~0.3 Gb, ~48,000 genes) (Healey et al. 2024). 224 \nIt must be noted that these results are likely affected by the availability of many more angiosperm 225 \ngenomes in public databases, while other plant and algal groups are not as well represented. As 226 \nmore genomes are sequenced, these analyses will have to be revisited.  227 \nWe also detected multiple occurrences of heterodimeric CAO genes, where the functional CAO 228 \nprotein is encoded by two genes (Table 1, Supplementary Table S1). Previous work in the 229 \nprasinophyte Micromonas pusila demonstrated that in this species CAO was encoded by two 230 \nseparate genes, one that possesses the mononuclear iron-binding motif (MpCAO1) and a second 231 \none that encodes for the Rieske cluster (MpCAO2). It was shown that a combination of both gene 232 \nproducts was necessary to form an active heterodimeric CAO protein (Kunugi et al. 2013; Dey et al. 233 \n2023). We expand on this work by showing the presence of a heterodimeric CAO encoded by two 234 \nseparate genes across the Mammiellophycea. This group within the prasinophytes contains M. 235 \npusila and several of its relatives (Worden et al. 2009), the ecologically important picoalgae 236 \nOstreococcus tauri, Ostreococcus lucimarinus and Bathycoccus prasinos (Palenik et al. 2007; 237 \nMoreau et al. 2012; Blanc-Mathieu et al. 2014), as well as the related Chloropicon primus 238 \n(Chloropicophyceae) (Lemieux et al. 2019).  239 \nWe also show that heterodimeric CAO is not restricted only within the Prasinophytes. We detect a 240 \nsimilar CAO gene arrangement in the genome of the multinucleate green alga Caulerpa lentillifera 241 \n(Bryopsidales) (Arimoto et al. 2019) , as well as in in the genome of Bigelowiella natans 242 \n(Chlorarachniophyceae) (Curtis et al. 2012). Phylogenetic analysis based on plastid-encoded 243 \nprotein sequences revealed that chlorarachniophytes acquired their secondary plastids by the 244 \nuptake of a filamentous green alga in the Bryopsidales (Suzuki et al. 2016). Thus, B. natans likely 245 \ngained a heterodimeric CAO from the Bryopsidales endophyte. Considering that CAO in all other 246 \ngreen lineages (Supplementary Table 1) and within the prochlorophytes (Nagata et al. 2004) is also 247 \nencoded by a single gene that contains both the Rieske and iron-binding domain  it is likely that the 248 \noccurrence of two separate CAO genes is not an ancestral condition. Instead, it has been 249 \npostulated  that CAO was encoded by a single gene in the common ancestor of green plants 250 \n(Kunugi et al. 2016). Indeed, it has been suggested that acquisition of two separate genes encoding 251 \nfor key functional domains may not be an evolutionarily difficult process. CAO likely functions as a 252 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n8 \n \ntrimer (Kunugi et al. 2013; Liu et al. 2022a), and the electron transfer from the Rieske to the 253 \nmononuclear iron occurs between two neighbouring CAO proteins. A functionally similar process 254 \nlikely occurs in the CAO enzyme encoded by two separate genes.  255 \nTable 1: Number of species across the Viridiplantae with fully sequenced and annotated genomes 256 \navailable through Phytozome (Total), compared to the number of species that encode for multiple 257 \nCAO genes (Multiple CAO) and number of species that encode for two CAO genes that form 258 \nheterodimeric complex (Heterodimeric CAO).  259 \n 260 \nGroup Total  Heterodimeric CAO Multiple CAO  \nChlorophytes* 70 11  8  \nCharophytes 10 0 2  \nNon-vascular plants 6 0 3  \nLycophytes 3 0 2  \nGymnosperms 1 0 0 \nAngiosperms  151 0 62  \n*The Chlorophyte group also includes Bigelowiella natans (Chlorarachniophyceae) that has gained 261 \nsecondary plastids by the uptake of a green alga. 262 \nAll examined streptophytes encode for at least one CAO gene (Supplementary Table 1) signifying 263 \nthe importance of Chl b for plant physiology, but we were not able to identify a homolog of CAO in 264 \nseveral chlorophyte algal species. Not surprisingly, we did not detect a CAO homolog in the 265 \ninvertebrate parasite Helicosporidium sp., a colorless protist that belongs within the 266 \ntrebouxiophycean green algal clade (Tartar et al. 2003). As a consequence of its parasitic lifestyle, 267 \nHelicosporidium has lost nearly all genes associated with light harvesting, photosynthesis and 268 \nchlorophyll biogenesis (Pombert et al. 2014), including CAO.   269 \nWe were also not able to conclusively identify a CAO gene in the genomes of the endolithic coral 270 \nholobiont Ostreobium quekettii (Bryopsidales) (Iha et al. 2021), the aquaculture species 271 \nTetraselmis striata (Chlorodendrophyceae) (Steadman Tyler et al. 2019) and the halotolerant 272 \nPicocystis sp. ML (Picocystophyceae) (Junkins et al. 2019). All identified sequences within the 273 \ngenomes of these species had e-values higher than the cut-off (≥e-15) used in this work and high 274 \nsimilarity to pheophorbide a oxygenase (PAO), a Rieske-type protein involved in chlorophyll 275 \ndegradation (Pružinská et al. 2003). The lack of CAO genes in these algae is surprising and could be 276 \nan artifact of genome assembly, as Chl b has been detected in all three species (Roesler et al. 277 \n2002; Massé et al. 2020; Conlon et al. 2024). The genome of O. queketti has a low BUSCO score 278 \n(60.7%) (Iha et al. 2021) and the BUSCO score is not reported for the draft genomes of T. striata and 279 \nP. sp. ML (Junkins et al. 2019; Steadman Tyler et al. 2019). The presence of CAO should be 280 \nexperimentally examined in these species. 281 \nIt is tempting to speculate that these species may have a different mechanism for Chl b 282 \nbiosynthesis or a CAO sequence significantly different than most algae and plants. For instance, 283 \nOstreobium is an endolithic coral holobiont adapted to extreme shading, and has lost many genes 284 \nassociated with photoprotection and light perception due to evolution in a light-limited 285 \nenvironment (Iha et al. 2021). This alga is also the only known eukaryote that has lost the gene 286 \nencoding light-dependent protochlorophyllide oxidoreductase (LPOR), a key enzyme that catalyzes 287 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n9 \n \nthe conversion of protochlorophyllide to chlorophyllide a, a necessary step towards the synthesis 288 \nof Chl a (Reinbothe et al. 2010). Instead, this alga fully depends on dark operative POR (DPOR) to 289 \nsynthesize Chl a, likely as an adaptation to extreme shading (Iha et al. 2021). The mechanism of 290 \nchlorophyll biosynthesis has not been examined in the species in detail, but it is clear that the 291 \nendolithic lifestyle has resulted in a unique physiology. Further work will shed light on the role of 292 \nCAO in this and other shade-tolerant species.  293 \nWe used a very conservative method to identify putative CAO sequences with both a Rieske and 294 \nFe-binding domains, but this strict approach may have prevented the identification of duplicates 295 \nthat may have diverged or become pseudogenes. Indeed, using a less conservative cutoff (≤e-5) 296 \nwhen screening the genomes of A. thaliana and C. reinhardtii (Wang et al. 2022; Craig et al. 2022) 297 \nreveals that PAO and Translocon at the inner envelope membrane of chloroplasts 55 (TIC55) may 298 \nbe paralogous to CAO (Supplementary Table S2). PAO and TIC55 encode for enzymes with a Rieske 299 \nand Fe-binding domains but share a very low sequence similarity with CAO and each other 300 \n(Supplementary Table S2, Supplementary Figure S1). Both PAO and TIC55  are involved in 301 \nchlorophyll breakdown and allow for the degradation of the highly phototoxic pheophorbide a and 302 \nits export from the chloroplast (recently reviewed in Kuai et al. 2018). Evolutionary analysis of the 303 \ngenes involved in chlorophyll degradation revealed that this pathway was already present in the 304 \ncommon ancestor of land plants, suggesting that PAO and TIC55 may share their origins with CAO 305 \n(Schumacher et al. 2022). This hypothesis has not yet been experimentally supported as ancestral 306 \nmonooxygenase have been difficult to distinguish without detailed knowledge on their biochemical 307 \nactivity and function (Schumacher et al. 2022). This possibility raises interesting questions about 308 \nthe ancient evolutionary origins of chlorophyll metabolism within the Viridiplantae.  309 \n Was CAO gene duplication an ancestral event?  310 \nTo determine whether the CAO gene duplication was an ancestral event, we performed a 311 \nphylogenetic analysis of all high-quality CAO amino acid sequences (except heterodimeric CAO). 312 \nPhylogenetic patterns typically match previously shown relationships among major Viridiplantae 313 \ngroups (Figure 2), although some relationships are poorly resolved (particularly among the algal 314 \ngroups and the Rosids; Supplementary Figure 2). The presence of two CAO genes has been 315 \npreviously reported in rice (OsCAO1 and OsCAO2) where the two genes were positioned in tandem 316 \non Chromosome 10, taken as evidence for a recent gene duplication in this species (Lee et al. 317 \n2005). A tandem CAO duplication, however, appears to be the exception rather than the rule. In 318 \naddition to rice, we only observed CAO duplicates located in proximity to each other in the 319 \ngenomes of a handful of species: the green alga Volvox reticuliferus, the lycophyte Diphastrium 320 \ncomplanatum, and the magnoliid Cinnamomum kanehirae (Table 2).  321 \nThere are several instances, mainly in very closely related species, where the duplication may have 322 \noriginated in a recent shared ancestor. There are several possible cases in the Trebouxiophyceae 323 \nand Chlorophyceae class, where CAO duplicates are detected in closely related Chlorella and 324 \nScenedesmus species (Supplementary Figure 2A). This insight must be interpreted with caution as 325 \nphylogenetic resolution within the chlorophytes is still weak due to limited gene and taxon 326 \nsampling (Li et al. 2021).  Within the bryophytes, Spaghnum magellanicum and Sphagnum falax, 327 \nspecies that share a very close evolutionary relationship (Piatkowski and Shaw 2019; Bell et al. 328 \n2020), both have two CAO genes possibly arising from an ancestral duplication. Two CAO genes 329 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n10 \n \nare also encoded in the genome of P. patens (Zhang et al. 2023). This raises the possibility of a CAO 330 \nduplication in a recent shared ancestor (Supplementary Figure 2B), but the bryophytes are 331 \nrepresented by only 5 species in our analysis. Additional genomic sequencing and a deeper 332 \nphylogenetic analysis will be needed to clarify the evolution of CAO within these groups.  333 \nFigure 2: A phylogenetic tree based on CAO amino acid sequences inferred from using maximum 334 \nlikelihood analysis, using 326 unique CAO sequences across 246 species color-coded to represent 335 \nthe major Viridiplantae groups. Scale bar indicates amino acid substitutions per site. Species with 336 \nmore than one detected CAO gene are highlighted in red 337 \n   nob ct ri \n h oro h t \n h ro h t \n r o h t \n  co h t \n   nos  r s\n   no ii s\n onocots\nAst ri s\n  s   osi s\n  bi s\n    i s\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n11 \n \nSpecies that encode for multiple CAO genes within their genomes are more prevalent among 338 \nangiosperms. Within the monocots, CAO duplicates arising from a shared ancestral gene are likely 339 \npresent in the Asparagaceae, specifically among the closely related Agave tequiliana, Yucca 340 \nfilamentosa and Yucca aloifolia (Ji et al. 2023) , although we were only able to detect a single CAO 341 \ncopy in Asparagus officinalis (Supplementary Figure 2C). We detected two CAO gene copies in two 342 \nBrachypodium species (B. mexicanum and B. hybridum) but not in their relatives from the same 343 \ngenus (B. arbuscula, B. stacei, B. sylvaticum and B. distachyon), raising the possibility of an 344 \nancestral duplication followed by gene loss. All phylogenetic studies in Brachypodium support a 345 \nrapid and recent divergence from a common ancestor (Catalan et al. 2016), supporting this 346 \npossibility.  347 \nCAO duplication may be ancestral in the two closely related Saxifragales Kalanchoe laxiflora and 348 \nKalanchoe fedtschenkoi (Han et al. 2024). We detected two CAO genes in K. fedtschenkoi and four 349 \nin K. laxiflora, possibly arising in a recent ancestor, followed by a second duplication within K. 350 \nlaxiflora (Supplementary Figure 2D). Within the Fabids, four Populus species and their relative Salix 351 \npurpurea, each encode for two CAO gene copies that may be a result of an ancestral duplication. 352 \nThe cultivated soybean Glycine max and its wild relative Glycine soja each encode for four CAO 353 \ngene copies (Supplementary Figure 2D) that are closely related to their homologs between the two 354 \nspecies. The phylogenetic separation of CAO1/2 and CAO3/4 from G. max and G. soja is puzzling, 355 \nas the two species are the closest relatives within the Glycine genus (Zhuang et al. 2022). All 356 \nGossypium species examined here encode for four CAO genes, except G. raimondii which encodes 357 \nfor two copies (Supplementary Figure 2E). The only other member of the Malvaceae examined, 358 \nTheobroma cacao, encodes for a single CAO gene, and although it clusters with CAO from 359 \nGossypium sp., the relationship is poorly supported. Finally, there is a possibility of ancestral 360 \nduplications within the Brassicaceae (Supplementary Figure 2E), but this family has a poor 361 \nresolution due to factors such as incomplete sampling, rapid radiation, and hybridization (Huang et 362 \nal. 2016; Hendriks et al. 2023).  363 \nThe genomes of multiple species across the Viridiplantae encode for more than one CAO gene, but 364 \nare these gene duplications related to plant evolutionary patterns? The green lineage is 365 \ncharacterized by a dynamic and complex evolutionary history marked by multiple whole genome 366 \n(WGD) and segmental duplication events, gene family expansions, and lineage-specific gene 367 \nlosses (Panchy et al. 2016; Kersey 2019; Leebens-Mack et al. 2019; Soltis and Soltis 2021).  Such 368 \ntremendous diversity in genome content can be a challenge for elucidating CAO evolutionary 369 \ntrajectories even withing a single family. For instance, the Brassicaceae have been shaped by 370 \nmultiple well-established WGD events (Mabry et al. 2020), but we detected only a single CAO gene 371 \nin the genome of A. thaliana and its closest relatives. In contrast, the genomes of several other 372 \nBrassica species encode for two or more CAO genes (Figure 2). This highlights the importance of 373 \nconsidering not only gene duplication but also gene retention dynamics when interpreting the 374 \nevolution of CAO. Future work that incorporates in-depth analyses, such as relative dating of CAO 375 \nduplication events and genome-wide synteny, will further clarify the evolutionary trajectory of CAO 376 \nwithin the Viridiplantae. 377 \nWe show that CAO gene duplication is not a rarity among the Viridiplantae, but are all CAO 378 \nduplicates expressed and do they encode for active proteins? In our work, we focused only on CAO 379 \ngenes with highly conserved catalytic domains likely to encode for active enzymes, but the 380 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n12 \n \nfunctional role of CAO duplication may be complex. A survey of published transcriptomes from 16 381 \nrepresentative species from all major groups (Chlorophytes, Charophytes, non-vascular and 382 \nvascular plants) suggests that all CAO duplicate genes within a species are expressed, albeit at 383 \ndifferent levels. Typically, one CAO gene has a higher expression than its paralog within the same 384 \ngenome (Supplementary Table 4) suggesting differential regulation of CAO expression.  385 \nSignificantly, all experimental studies to date suggest that the expression of CAO duplicates may 386 \nbe differently regulated by light availability. In rice, the expression of OsCAO1 is light-inducible, 387 \nwhile OsCAO2 accumulates during periods of darkness and is expressed mainly in non-388 \nphotosynthetic tissues (Lee et al. 2005). In P. patens, both PpCAO1 and PpCAO2 are expressed in 389 \nboth light and dark conditions, but only PpCAO1 is upregulated with prolonged light exposure. 390 \nSimilarly, in two Chlamydomonas species that harbour CAO duplicate genes (Chlamydomonas 391 \npriscui and Chlamydomonas sp. ICE-MDV), only one CAO homolog is upregulated in response to 392 \nincreases in light intensity, while the second one is constitutively expressed (Poirier et al. 2025). 393 \nThe role of light in regulating CAO expression has been also demonstrated previously in 394 \nArabidopsis and tobacco, species with a single CAO gene copy (Pattanayak et al. 2005; Tanaka and 395 \nTanaka 2005; Biswal et al. 2012, 2024).   396 \nAdditionally, the mechanisms behind the control of Chl b synthesis may be species specific. 397 \nMutational analysis in rice revealed that only OsCAO1 was necessary for Chl b biosynthesis. 398 \nOsCAO1 knockout mutants exhibited low Chl b levels, disordered thylakoid membrane 399 \narrangements, membrane damage due to excessive ROS accumulation and decreased chilling 400 \ntolerance (Lee et al. 2005; Jung et al. 2021; Xiong et al. 2024), but these effects were absent in 401 \nOsCAO2 knockouts (Lee et al. 2005). In contrast, knocking out either of the two CAO homologs in 402 \nP. patens lead to significantly reduced Chl b levels suggesting a dependency on two active CAO 403 \nenzymes for the appropriate control of Chl b synthesis in this species (Zhang et al. 2023).  404 \nIt is tempting to speculate that species that encode for multiple CAO genes have a more robust 405 \ncapacity for Chl b biosynthesis through an increase in gene dosage or a finer control over Chl b 406 \naccumulation though gene subfunctionalization or differential expression (Kondrashov 2012; 407 \nPanchy et al. 2016). Two gene copies with different expression patterns or activity levels could 408 \nprovide a fine-tuned control of Chl b levels and photosynthetic performance under varying 409 \nenvironmental conditions, but additional experimental work is needed to substantiate this 410 \nhypothesis. 411 \n 412 \n 413 \n 414 \nTable 2: Distribution of CAO duplicate genes across Viridiplantae. Only genes with a confirmed 415 \nRiske conserved cluster, Fe-binding domain (all species), and degron sequence (Streptophytes 416 \nonly) are listed. A checkmark (✓) indicates that the CAO genes are positioned in a tandem on the 417 \nsame chromosome within the genome, while a cross (x) indicates the genes are located on 418 \ndifferent chromosomes. Accession numbers can be found in Supplementary Table 1 (continued on 419 \nnext page). 420 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n13 \n \nTaxon Species #CAO genes Tandem \nChlorophyta    \nChlorophyceae Chlamydomonas priscui 2 x \nChlorophyceae Volvox reticuilferus 2 ✓ \nChlorophyceae Tetradesmus deserticola 2 x \nTrebouxiophyceae Chlorella sp. A99 2 x \nTrebouxiophyceae Chlorella sorokiniana 2 x \nTrebouxiophyceae Chlorellaceae sp.  2 x \nTrebouxiophyceae Micractinium conductrix 2 x \nPedinophyceae Pedinomonas minor 2 x \nCharophyta  \n  \nZygnematophyceae Mesotaenium endlicherianum 2 x \nZygnematophyceae Spirogloea muscicola 2 x \nBryophyta  \n  \nBryopsida Physcomitrium patens  2 x \nSphagnopsida Sphagnum fallax  2 x \nSphagnopsida Sphagnum magellanicum 3 x \nLycophyta  \n  \nEquisetopsida Diphasiastrum complanatum  4 ✓ \nEquisetopsida Ceratopteris richardii  2 x \nAngiosperms  \n  \nLaurales Cinnamomum kanehirae 2 ✓ \nNymphaeales Nymphaea colorata 2 x \nAsparagales Agave tequilana  2 x \nAsparagales Yucca aloifolia  3 x \nAsparagales Yucca filamentosa 3 x \nZingiberales Musa acuminata v1 2 x \nAlismatales Spirodela polyrhiza v2 2 x \nPoales Typha latifolia  2 x \nPoales Eleusine coracana  2 x \nPoales Oryza sativa  2 ✓ \nPoales Thinopyrum intermedium  3 x \nPoales Brachypodium hybridum 2 x \nPoales Brachypodium mexicanum  2 x \nPoales Zea mays  2 x \nPoales Andropogon gerardi  3 x \nPoales Miscanthus sinensis  2 x \nPoales Panicum virgatum  2 x \nPoales Saccharum officinarum 6 x \nCaryophyllales Chenopodium quinoa  2 x \nGentianales Coffea arabica geisha  2 x \n \n \n  \n  \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n14 \n \n \nTable 2:(continued) \nTaxon Species #CAO genes Tandem \nApiales Hydrocotyle leucocephala 2 x \nBoraginales Ehretia anacua  2 x \nAsterales Helianthus annuus 2 x \nLamiales Olea europaea 2 x \nSaxifragales Kalanchoe fedtschenkoi 2 x \nSaxifragales Kalanchoe laxiflora 4 x \nFabales Arachis hypogaea 2 x \nFabales Chamaecrista fasciculata 2 x \nFabales Glycine max  4 x \nFabales Glycine soja 4 x \nFabales Lupinus albus 2 x \nFabales Phaseolus vulgaris  2 x \nFagales Carya illinoinensis 2 x \nRosales Fragaria x ananassa 3 x \nRosales Malus domestica  2 x \nMalpighiales Linum usitatissimum  4 x \nMalpighiales Populus deltoides  2 x \nMalpighiales Populus nigra x maximowiczii  2 x \nMalpighiales Populus tremula x alba  2 x \nMalpighiales Populus trichocarpa  2 x \nMalpighiales Salix purpurea  2 x \nSapindales Anacardium occidentale  3 x \nMalvales Gossypium barbadense  4 x \nMalvales Gossypium darwinii  4 x \nMalvales Gossypium hirsutum  4 x \nMalvales Gossypium mustelinum  4 x \nMalvales Gossypium raimondii  2 x \nMalvales Gossypium tomentosum  4 x \nBrassicales Alyssum linifolium  2 x \nBrassicales Caulanthus amplexicaulis 2 x \nBrassicales Iberis amara  2 x \nBrassicales Lepidium sativum  2 x \nBrassicales Stanleya pinnata 2 x \nBrassicales Brassica juncea  3 x \nBrassicales Brassica oleracea 2 x \nBrassicales Cakile maritima 2 x \nBrassicales Crambe hispanica 2 x \nBrassicales Eruca vesicaria  2 x \nBrassicales Sinapis alba  3 x \nBrassicales Camelina sativa  3 x \nBrassicales Isatis tinctoria 4 x \n 421 \n 422 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n15 \n \nEvolutionary implications of the CAO degron sequence 423 \nIn addition to light-dependant regulation at the level of transcription, the main regulation of CAO 424 \noccurs post-translationally. Early work in A. thaliana demonstrated that this regulation is governed 425 \nby the presence of a degron sequence (QDLLTIMILH) in the protein’s N-terminal A domain that acts 426 \nas a docking site for a chloroplast Clp protease (Yamasato et al. 2005; Nakagawara et al. 2007; 427 \nSakuraba et al. 2009). It has been hypothesized that the degron sequence is “hidden” within the A 428 \ndomain when there are insufficient amounts of Chl b, but accumulation of this product causes a 429 \nconformational change in the CAO protein, exposing the degron, and giving access to the protease 430 \n(Tanaka and Tanaka 2011). The evolution of this negative-feedback regulatory loop has not been 431 \nexamined in detail, but it has been suggested that the degron was acquired early in the 432 \nViridiplantae evolution as a mechanism for high light adaptation (Kunugi et al. 2016).   433 \nHere we show that the degron sequence is highly conserved among all land plants (86% identity), 434 \nmoderately conserved among charophytes and non-vascular plants (68% and 66% identity 435 \nrespectively), and very poorly conserved in chlorophytes (39% identity) (Figure 3; Supplementary 436 \nFigure 3). Land plants (both vascular and non-vascular) have several highly conserved sites across 437 \nall species (Asparagine at position 2, Isoleucine at position 6, Histidine at position 10), whereas 438 \nthese positions are only moderately conserved in charophytes, and poorly conserved in 439 \nchlorophytes. In contrast, the Rieske cluster [CXH(X)15-17CXXH] exhibits an 84% pairwise identity 440 \namong all species, where the two histidine (positions 3 and 23) and cysteines (positions 1 and 20) 441 \nthat coordinate the metallocentre [2Fe-2S] (Przybyla-Toscano et al. 2021) are present in all 442 \nexamined sequences. Nearly all residues are conserved among the Viridiplantae in the C-terminal 443 \nFe-binding domain (96% pairwise identity). Overall, these results suggest that the mechanism of 444 \nChl b biosynthesis involving the Rieske and Fe-binding domains is ancestral and conserved among 445 \nall photosynthetic species, but this is not the case for the regulatory degron domain.  446 \nThe lack of a highly conserved degron sequence may have several implications in the evolution of 447 \nefficient light harvesting. For instance, the N-regulatory domain (including the degron) is 448 \ncompletely absent in marine eukaryotic Mammiellophycea that encode for a heterodimeric CAO 449 \ngene, as well as in their deep-water relatives from the order Palmophyllales. These species 450 \ntypically have low Chl a/b ratios and, unlike land plants, can incorporate Chl b into their core 451 \nantennae. This was interpreted as a necessary adaptation for efficient light harvesting in deep 452 \nmarine environments characterized by low levels of blue-green light (Kunugi et al. 2016).  453 \n 454 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n16 \n \nFigure 3: Key domains in the CAO protein across the Viridiplantae. (A) Sequence logos of the 455 \nconserved motifs identified in the CAO protein sequences. N and C represent the N-terminus and 456 \nC-terminus, respectively. All sequence logos are superimposed on a graphical representation of 457 \nplant CAO sequences (not to scale) showing the regulatory degron motif, conserved Rieske domain 458 \ncluster and Fe-binding domain (Sakuraba et al., 2009; Liu et al., 2022). The logos were generated 459 \nfor CAO proteins from chlorophyte algae, charophyte algae, non-vascular plants, and vascular 460 \nplants. The sequences on top of each logo are the consensus sequence with the most common 461 \nbase in each sequence alignment. (B) Comparison of the CAO amino acid sequences 462 \ncorresponding to the degron and its surrounding region in the N-terminal regulatory domain in 463 \nrepresentative species from Chlorophyte, Charophyte, non-vascular and vascular land plants. The 464 \nregion that corresponds to the degron sequence as described in A. thaliana is emphasized with a 465 \nbox. Amino acids that are identical with the A. thalina sequence are colored, and those that are 466 \ndifferent are shown in white.  467 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n17 \n \nIt has been suggested that the degron sequence evolved in shallow-water algae and land plants as 468 \na means to regulate Chl b accumulation and limit Chl b incorporation into the reaction center in an 469 \nenvironment characterized with high light intensities (Kunugi et al. 2016). Indeed, such species 470 \nexclusively harbour Chl a in their core complexes, but this appears to be controlled by pigment 471 \navailability rather than the chemical nature of the two chlorophyll types. The binding affinity of Chl 472 \na and b to the PSI complex is similar (Ikegami et al. 2007) and cyanobacteria that do not naturally 473 \nproduce Chl b successfully incorporated this pigment into their reaction centers when transformed 474 \nwith plant CAO (Satoh et al. 2001; Xu et al. 2001). Incorporation of Chl b into the reaction center 475 \naccompanied by increased Chl b accumulation was also observed in Arabidopsis when full length 476 \nCAO was overexpressed or replaced with a prokaryotic CAO that lacked a degron sequence 477 \n(Hirashima et al. 2006; Sakuraba et al. 2009).  478 \nThe chlorophyll cycle has not been examined in detail in the core chlorophytes (Chlorophyceae, 479 \nTrebouxiophyceae, Ulvophyceae), a diverse group that inhabits marine, freshwater and terrestrial 480 \nhabitats (Leliaert et al. 2012). Although many chlorophytes thrive in high light, others are adapted to 481 \nlight limited environments, including deep waters. As a result, these species have developed 482 \nadaptations allowing for efficient light harvesting in low light. For instance, Chlamydomonas priscui 483 \nis endemic to the depths of the Antarctic Lake Bonney, an environment characterized by extreme 484 \nshading and dominated by blue-green wavelengths (Neale and Priscu 1995). This alga displays a 485 \nhost of adaptations to extreme shading including a large LHC antennae size (reviewed in 486 \nCvetkovska et al. 2017; Hüner et al. 2022). As suggested for marine prasinophytes (Kunugi et al. 487 \n2016), such low-light adapted chlorophytes could benefit from increased Chl b levels and could 488 \nthrive without a Chl b-dependent feedback mechanism.  489 \nDespite the lack of a conserved degron sequence to regulate CAO protein levels (Figure 3), 490 \nchlorophytes have maintained the ability to alter their Chl a/b ratio in response to light (Leliaert et 491 \nal. 2012). Transcriptional control of CAO levels was postulated to be a minor aspect of adjusting 492 \nChl b levels in plants (Tanaka and Tanaka 2019), but this process could play a bigger role in 493 \nchlorophytes. It is also possible that a different, yet unidentified, mechanism exists for Chl b 494 \nturnover independent of a degron sequence, allowing these species to modify their Chl a/b ratios. 495 \nFinally, we can not rule out that the region corresponding to a degron in Chlorophyte CAO may still 496 \nhave a similar function despite poor sequence conservation with land plants. The mechanism of 497 \nregulating protein degradation via degron motifs is well understood in plants (Isono et al. 2024), but 498 \ninsights on algal degrons is lacking.  A detailed experimental investigation on the control and 499 \nmechanism of Chl b biosynthesis is clearly needed among the Chlorophytes. For instance, targeted 500 \nmutagenesis of the N-terminus of algal CAO will reveal the importance and role of this regulatory 501 \nregion in chlorophyll turnover.  502 \nConclusion and Drawbacks 503 \nOur work suggests that CAO gene duplication is widespread among the Viridiplantae, likely 504 \noriginating from multiple independent duplication events throughout plant evolution. While we 505 \nshow that the presence of multiple CAO gene copies is highly prevalent in land plants, these 506 \nresults must be interpreted with caution. Many more angiosperm genomes are available in public 507 \ndatabases, while other plant and algal groups are not as well represented. Thus, the conclusions of 508 \nour study must be re-visited once more genomes are sequenced. Furthermore, many genome 509 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n18 \n \nsequencing initiatives are focused on plants of economic and agricultural importance, in which 510 \ndomestication and selective breeding may have affected the genomic content (Turner-Hissong et 511 \nal. 2020). While the exact physiological impact of CAO duplication is unknown, a study in 512 \ntransgenic tobacco overexpressing CAO (Biswal et al. 2012) showed that an increase in Chl b may 513 \nbe a strategy to enhance CO2 assimilation, delay senescence, and enhance productivity. Such 514 \ntraits would be attractive in crop breeding programs (Voitsekhovskaja and Tyutereva 2015), and the 515 \nhigh prevalence of CAO duplication seen in crop plants in this study may have been selected for 516 \nduring plant breeding. Indeed, several crop plants (including corn, soybean, and olives, among 517 \nothers) appear to express all CAO genes encoded in their genome (Supplementary Table 4). The 518 \nfunctional role of these duplicates in plant physiology, photosynthetic efficiency, and productivity 519 \nwarrants further investigation.  520 \nOur understanding of the prevalence and evolution of CAO at the genetic level will be improved in 521 \nfuture years, as more algal and non-vascular plant genomes become available. Furthermore, there 522 \nare still numerous complex and outstanding questions regarding the control of Chl b accumulation 523 \nin photosynthetic species. In particular, the chlorophytes are an understudied group when it 524 \ncomes to Chl b regulating mechanism. Detailed genomic and experimental studies will shed light 525 \non the role and regulation of Chl b in these species and will deepen our understanding on this 526 \ncrucial photosynthetic pigment.  527 \nSupplementary Information 528 \nSupplementary Table S1: A summary of genes encoding for chlorophyllide a oxygenase (CAO) 529 \nacross the Viridiplantae used in this work. All sequences were obtained from Phycocosm 530 \n(Chlorophytes, Euglenophytes, Cercozoa, Charophytes) or Phytozome v13 (Bryophytes, 531 \nMarchantiophytes, Lycophytes, Gymnosperms, Angiosperms). Accession numbers represent the 532 \nunique IDs assigned to each gene (Phycocosm: Gene portal|Protein ID|Gene Model; Phytozome: 533 \nGene Model). E-values are based on a tBLASTn search with the Chlamydomonas reinhardtii CAO 534 \n(Cre01.g043350) for species within the Chlorophyta and Charophyta, and the Arabidopsis thaliana 535 \nCAO (AT1G44446) for all other species 536 \nSupplementary Table S2: BLAST results obtained by screening the genome of Arabidopsis thaliana 537 \nand Chlamydomonas reinhardtii for CAO homologs. Accession numbers were obtained from 538 \nPhytozome v13 (Goodstein et al. 2012). The functional domains according to InterPro (Blum et al. 539 \n2025) that are shared among all proteins are highlighted with an asterisk (Rieske Domain; InterPro: 540 \nIPR017941; Pheophorbide a Oxygenase Domain; InterPro: IPR013626).  541 \nSupplementary Table S3: A summary of the occurrence of CAO duplicate genes across the major 542 \ngroups within Viridiplantae. The number in brackets represents the proportion of species which 543 \nencode for multiple copies of the CAO gene (Mutiple CAO) or encode for two CAO genes that form 544 \nheterodimeric complex (Heterodimeric CAO). 545 \nSupplementary Table S4: CAO gene expression in species whose genome encodes for more than 546 \none CAO gene. All data was obtained from previously published work. Gene expression is 547 \npresented as FPKM, RPKM or TPM averages with standard deviation. In all cases, gene expression 548 \nratios were determined by dividing all expression values by the highest CAO expression level within 549 \nthe species. 550 \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted August 18, 2025. ; https://doi.org/10.1101/2025.02.10.637544doi: bioRxiv preprint \n\n19 \n \nSupplementary Figure 1: An alignment of the CAO protein from (A) Arabidopsis thaliana and (B) 551 \nChlamydomonas reinhardtii with all other proteins identified using the CAO amino acid sequence 552 \nas a query. The color of the residue depends on the degree of conservation among proteins (blue 553 \nfully conserved; green 80–100% similar; yellow 60–80% similar; white < 60% similar). The important 554 \ndomains that are shared among all proteins are highlighted in green (Rieske Domain; InterPro: 555 \nIPR017941), yellow (Fe-binding domain) and blue (Pheophorbide a Oxygenase Domain; InterPro: 556 \nIPR013626).  557 \nSupplementary Figure 2: A phylogenetic tree of CAO genes inferred from using maximum 558 \nlikelihood analysis. The node labels show the bootstrap support, with dashed lines indicating <50 559 \nbootstrap support. Scale bar indicates amino acid substitutions per site. Species with more than 560 \none detected CAO gene are highlighted in red. Cases where CAO duplication may have arisen in a 561 \ncommon shared ancestor are marked with a blue arrow, and only those located on branches with a 562 \nstrong bootstrap support (>85%) are shown. The inset shows the outline of the full CAO 563 \nphylogenetic tree inferred from using maximum likelihood analysis, using 326 unique CAO 564 \nsequences across 246 species color coded to represent the major Viridiplantae groups. The 565 \nposition of the chlorophytes (green) is highlighted with a black box. (A) Chlorophytes; (B) 566 \nCharophytes, bryophytes (including marachantiophytes), and lycophytes; (C) Gymnosperms, 567 \nmagnoliids, and monocots; (D) Basal eudicots, asterids and fabids; (F) Malvids. 568 \nSupplementary Figure 3: Multiple sequence alignment of the predicted CAO amino acid 569 \nsequences from Chlorophytes (A), Charophytes (B), non-vascular plants (C) and vascular plants 570 \n(D). Global alignments were performed with MUSCLE. The colour of the residue depends on the 571 \ndegree of conservation among species (blue, fully conserved; green, 80–100% similar; yellow, 60–572 \n80% similar; gray, < 60% similar). 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