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Mannitol biosynthesis in algae: more widespread and diverse than previously thought

2016; Wiley; Volume: 213; Issue: 4 Linguagem: Inglês

10.1111/nph.14358

1469-8137

Thierry Tonon, Yongguang Li, Simon J. McQueen‐Mason,

Algal biology and biofuel production

Mannitol is probably the most abundant naturally occurring polyol. It is produced by a wide range of living organisms, with the noticeable exception of the Archaea and the animal kingdom. It fulfils key physiological roles, including carbon storage and protection against environmental stress (Iwamoto & Shiraiwa, 2005; Patel & Williamson, 2016). Indeed, mannitol can act as organic osmolyte, compatible solute, anti-oxidant, or thermal protectant. Among Eukaryotes, plants rely on mannose-6-phosphate reductase for mannitol production from fructose-6-phosphate (F6P) (Stoop et al., 1996). Apicomplexa (Schmatz et al., 1989) and algae (Karsten et al., 1997) have evolved a different pathway, where mannitol-1-phosphate dehydrogenase (M1PDH) reduces F6P into mannitol-1-phosphate (M1P), which is then hydrolysed to mannitol by a mannitol-1-phosphatase (M1Pase) (Fig. 1a). A similar pathway occurs in fungi (Solomon et al., 2007). In algae and Apicomplexa, both reactions are part of the mannitol cycle supporting production and recycling of this polyol. Genes and proteins for M1PDH have been characterized from fungi, but not for M1Pase. Conversely, both M1PDH and M1Pase genes and enzymes have been studied in the Apicomplexa Eimeria tenella (Schmatz et al., 1989; Liberator et al., 1998). In algae, mannitol occurs in several lineages (Supporting Information Table S1) and can represent up to 25% of their dry matter (Reed et al., 1985). Both M1PDH and M1Pase activities have been determined in the red microalga Dixoniella grisea (Eggert et al., 2006), the macroalga Caloglossa leprieurii (Karsten et al., 1997), and endogenous enzymes have been purified from Caloglossa (Iwamoto et al., 2001, 2003), but the encoding genes have not been identified yet. M1PDH activity has also been measured in the green alga Platymonas subcordiformis (Richter & Kirst, 1987). Mannitol metabolism has been well studied in brown algae, and recombinant Ectocarpus M1PDH and M1Pase both characterized (Rousvoal et al., 2011; Groisillier et al., 2014; Bonin et al., 2015). M1PDH phylogenetic analysis indicated that bacterial/fungal and apicomplexa/algal sequences formed two distinct groups among the polyol specific long chain dehydrogenase/reductase family (PSLDR) (Bonin et al., 2015). Moreover, Ectocarpus and Eimeria M1Pases belong to distinct families of phosphatases: the haloacid dehalogenases (HAD-M1Pase) for the former, and the histidine phosphatases (His-M1Pase) for the latter (Liberator et al., 1998; Groisillier et al., 2014). Algae represent a polyphyletic group with differing life styles (aquatic, terrestrial, extremophile, symbiotic), and their importance to aquatic ecosystems and global carbon balances makes them an important area of study (Field et al., 1998). Algae arose from a complex evolutionary history involving endosymbioses and lateral gene transfers (LGTs) that have shaped their metabolic networks (Falkowski et al., 2004). In this context, it was first suggested that brown algal M1PDH and M1Pase genes were acquired by LGT from actinobacteria (Michel et al., 2010). Subsequently, it was proposed that these genes were transferred from bacteria to an ancestral Ochrophyta after the separation of diatoms (Dittami et al., 2011). To increase understanding of mannitol biosynthesis in algae, we had two objectives for this study: (1) to assess the occurrence of enzymes involved in mannitol biosynthesis across algal lineages, based on previously characterized algal and apicomplexa M1PDHs and M1Pases; and (2) to analyse the evolution of the mannitol biosynthetic pathway in these organisms. Mining of recently available transcriptomic and genomic resources revealed an unexpected diversity of M1PDHs and M1Pases, and we put forward hypotheses for their evolution among algae. Algal M1PDH and M1Pases were identified using homology searches to the protein sequences of biochemically characterized M1PDH (ES0017G00030_Esil) and HAD-M1Pase (Es0100G00180_Esil) of the brown alga Ectocarpus (Groisillier et al., 2014; Bonin et al., 2015), and His-M1Pase (AF032462_Eten) of the apicomplexa Eimeria tenella (Liberator et al., 1998). These sequences were used to interrogate the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP, http://marinemicroeukaryotes.org/), the 1000 plants transcriptome project (OneKP; https://sites.google.com/a/ualberta.ca/onekp/), the Eukaryotic Pathogen Database Resources (EuPathDB; http://eupathdb.org/eupathdb/), and proteomes translated from individual algal genomes (Table S2). Hit sequences with E-value ≤ 1E-05 were collected for further analysis (Notes S1–S4). The molecular evolutionary genetics analysis 6 (Mega6; Tamura et al., 2013) software was used for multiple sequence alignments, performed with Muscle, and for phylogenetic analysis using bootstrapped maximum likelihood. Signal peptide and sub-cellular localization were predicted using PredAlgo (Tardif et al., 2012), ASAFind (Gruber et al., 2015), and PlasmoAP (Foth et al., 2003). An expanded version of the Materials and Methods section is given in Methods S1. When analysing these results, it is important to consider that the abundance of particular transcripts in transcriptomes is influenced by culture conditions and life cycle stages, and that nonoccurrence in the transcriptome does not necessarily equate to nonoccurrence in the genome of the corresponding organism. Similarly, it is possible that absence of sequences in genomes may result from insufficient depth of sequencing and/or the absence of prediction for genes of interest. For algae resulting from primary endosymbiosis (Fig. 1b), mannitol has been reported in some Chlorophyta, in a few species of Rhodophyta, but not in Charophyta and Glaucophyta. In accordance with these observations, our studies revealed no M1PDH or M1Pase genes in Charophyta or Glaucophyta species analysed (Fig. 1b; Table S3). However, such genes are present in a small number of species of Chlorodendrophyceae, Mamiellophyceae, and Pyramimonadophyceae, and in one Rhodophyta species. Interestingly, the types of mannitol biosynthetic genes seen in these species are generally diverse and parallel pathways seem to exist in many of them. All Mamiellophyceae species contain a bi-functional M1PDH/HAD-M1Pase fusion protein alongside a standalone HAD-M1Pase (stHAD-M1Pase). Two distinct pathways appear to be available in species of Tetraselmis (Chlorodendrophyceae) and Pyramimonas (Pyramimonadophyceae) investigated, one based on standalone M1PDHs (stM1PDHs) and stHAD-M1Pases, the other on bi-functional proteins. A similar pattern is apparent in the Rhodophyta Rhodella maculata. No genomic or transcriptomic resources were available to assess the occurrence of M1PDH and M1Pase in the only Floridophyceae known to produce mannitol, i.e. species of the genus Caloglossa (Karsten & West, 1993). Mannitol biosynthetic genes are also identified in algae resulting from green-algal secondary endosymbiosis (Fig. 1b). Euglenozoa, as in Mamiellophyceae, possess a stHAD-M1Pase and a bi-modular M1PDH/HAD-M1Pase, whilst Chlorarachniophyceae contain several copies of stM1PDH and stHAD-M1Pase genes. Among algae derived from red-algal secondary endosymbiosis (Fig. 1b), no genes of interest are observed in Cryptophyte species that were examined. In Haptophyceae, only some species of Prymnesiophyceae possess M1PDH and M1Pase genes, with two distinct profiles. While stHAD-M1Pases and standalone His-M1Pases (stHis-M1Pases) co-occur in Isochrysidales, only stHis-M1Pases have been identified in others, alongside at least one gene encoding a stM1PDH. In unicellular Ochrophyta, mannitol production has been reported in several classes, notably Bacillariophyceae (species Thalassiosira fluviatilis; Hellebust, 1965) and Chrysophyceae (species Ochromonas minima; Dittami et al., 2011). However, both species were not represented in datasets analysed. Meanwhile, genes of interest are absent in all Bacillariophyceae, Chrysophyceae and Bolidophyceae inspected, and are heterogeneously distributed in other classes. Only some Dictyochophyceae species possess a bi-functional M1PDH/HAD-M1Pase. Pelagophyceae and Pinguiophyceae contain stM1PDHs and stHis-M1Pases, while Eustigmatophyceae, Raphidophyceae, Phaeophyceae, and Xanthophyceae possess stM1PDHs and stHAD-M1Pases. Among Alveolates, mannitol has been reported in Amphidinium cartereae (Bidwell, 1957), but no mannitol biosynthetic genes were identified in this species. However, the corresponding genes are found in Chromerida, Apicomplexa of the Eimeria genus, and a minority of Dinoflagellates species examined. In these latter, a patchy distribution of M1PDH and M1Pase genes is observed, with several combinations of enzymes, including the occurrence of bi-functional proteins. A phylogenetic tree of M1PDH standalone proteins and modules was obtained after alignment of the sequences (Fig. S1; Notes S5), and it features several well-supported groups (Fig. S2). One illustrates the monophyly of green algal standalone enzymes and module sequences, except for the module identified in Pterosperma (Pyramimonadophyceae). Mamiellophyceae M1PDH modules form a sub-group distinct from the one containing Pyramimonas and Tetraselmis sequences. In Chlorarachniophyceae, all stM1PDHs are derived from a common ancestor. Conversely, at least two groups of standalone sequences are observed for some lineages resulting from red-algal secondary endosymbiosis, i.e. Haptophyceae, Raphidophyceae, Pelagophyceae, Xanthophyceae, and Phaeophyceae. However, there is no strong bootstrap support at the deeper branches for these lineages. In Dictyochophyceae, standalone and module M1PDHs are contained in separated groups. No trend for predicted localization/signal peptide can be inferred for most of these lineages (Table S4; Notes S5). However, 13 out of 20 of the brown algal group 1 sequences are predicted to be plastidial or to contain a signal peptide, while only three out of 16 for group 2 sequences show such signal sequences. A third type of M1PDH is observed in Ectocarpales, probably from duplication of a group 1 sequence, and in Pelagophyceae. Alveolate M1PDHs form several distinct sub-groups. Two types of standalones are distinguished in Chromerida. The cluster of Alexandrium standalones is closely related to two Pinguiophyceae homologues. Standalones and modules from other Dinoflagellates are part of the same sub-group, closely related to Pelagophyceae and Haptophyceae group 1 standalones. Analysis of HAD-M1Pases (Figs S3, S4; Notes S5) shows that modules and standalone proteins of Mamiellophyceae form two separate groups. Most Mamiellophyceae stHAD-M1Pases are predicted to be chloroplastic, in contrast to Tetraselmis and Pyramimonas sequences (Table S4; Notes S5). Chlorarachniophyceae stHAD-M1Pases are comprised of two independent groups, and an additional small cluster probably corresponds to a specific duplication in Bigelowiella species. As for M1PDHs, no specific trend is found in the predicted subcellular localization/presence of signal peptide in these M1Pases. Sequences from Eutreptiella and Rhodella form two distinct well-supported sub-groups, similar to the M1PDH phylogeny. In brown algae, two distinctive groups of stHAD-M1Pases are identified. Seven of the 20 sequences of group 1 is predicted to be plastidial, whereas none of the 19 in group 2 is. Raphidophyceae sequences occur in two sub-groups in which both Heterosigma akashiwo and Chattonella subsalsa are represented. Dinoflagellate standalones and modules are probably monophyletic because all are contained in one moderately supported group, closely related to the cluster formed by Prymnesiophyceae sequences. His-M1Pases were identified in all phyla derived from a red-algal endosymbiont, i.e. Haptophyceae, Ochrophyta, Chromerida, Dinoflagellates and Apicomplexa. Some sub-groups corresponding to these lineages are well supported (Figs S5, S6; Notes S5), such as Dictyochophyceae, Pelagophyceae and Alexandrium. The phylogenetic tree built for bifunctional M1PDH/M1Pases is robust, consisting of three main groups (Figs 2, S7; Notes S5). One supports the monophyly of the green algal sequences, except for Pterosperma. Moreover, Mamiellophyceae, Tetraselmis and Pyramimonas sequences belong to three distinct sub-groups, apart for the sequence 0118958248_Pobo for which the location in the tree is uncertain. Most of these green algal sequences are predicted to be plastidial. A second cluster groups together Eutreptiella, red algal, and Dinoflagellate sequences. Finally, a third group contains the three Dictyochophyceae and the Pterosperma fusions. Previous assumptions on evolution of mannitol biosynthesis in algae (Michel et al., 2010; Dittami et al., 2011) were revisited by analysis of extended transcriptomic and genomic resources. M1PDH and M1Pases genes are identified in most of the phyla analysed (Fig. 3), with different combinations of standalone and fusion proteins, in good correlation with the occurrence of mannitol in algae. Algal mannitol biosynthesis was probably shaped by the occurrence of new substrate specificity within the PSLDR, HAD, and histidine phosphatase superfamilies of proteins. Since extant cyanobacteria are not able to produce mannitol, we suggest that standalone M1PDH and HAD-M1Pase genes may have been present in the nonphotosynthetic eukaryotic host cell involved in primary endosymbiosis. This is also probably the case for one or several of the eukaryotic hosts involved in secondary and/or possible subsequent endosymbioses (Burki et al., 2016). Little information is available about the metabolic repertoire of these heterotrophic hosts (Gould et al., 2008) and their phylogenetic relationships remain unclear (Baurain et al., 2010). Mannitol biosynthetic genes were then lost in Glaucophyta, Charophyta, Rhodophyta, Cryptophyta, and Bacillariophyceae, retained as standalone proteins, or fused to create bi-functional proteins. Several specific duplication events occurred later on in several lineages. This concerns for instance M1PDHs and HAD-M1Pases in Chlorarachniophyceae, Tetraselmis and Pyramimonas species, notably for the occurrence of fusion proteins in these green algae. We also suggest that His-M1Pase may have been present in ancestors of the CASH lineages (Cryptophyta, Alveolata, Stramenopiles and Haptophyta; Baurain et al., 2010), and then subsequently lost in some individual genus/species. Moreover, LGTs between eukaryotic algae may have also contributed to the uneven distribution of M1PDHs and M1Pases in these organisms. Bi-modular M1PDH/M1Pases appear independently in different lineages. Such proteins in the green alga Pterosperma and in the red microalga Rhodella maculata represent interesting cases. The former probably arose by fusion of M1PDH and His-M1Pase modules acquired possibly by LGT(s) from Pelagophycae and/or a Dictyochophyceae, both classes being closely phylogenetically related (Brown & Sorhannus, 2010). Mannitol production is limited to a few Rhodophyta species, and identification of M1PDH and M1Pase in the macroalga Caloglossa should help to better understand evolution of this metabolic pathway in these organisms. Identification of mannitol biosynthetic genes shows that several pathways can co-exist in some algae, such as [stM1PDH+stHAD-M1Pase] and [stM1PDH+stHis-M1Pase], or [stM1PDH+stHAD-M1Pase] and bi-functional M1PDH/HAD-M1Pase, or even all three in some Dictyochophyceae. Furthermore, some species contain several genes for the same enzymatic activity, sometimes with different predicted cellular localization. Such apparent functional redundancy may be of physiological importance, and suggests intriguing questions about evolution, subcellular localization, regulation, and the ecological significance of algal mannitol synthesis pathways. This is particularly interesting in the era of 'Oceans Systems Biology' (Karsenty, 2012) and the study of phytoplankton given their major contributions to carbon cycling at the planetary scale (Bork et al., 2015). The authors thank Dr Erwan Corre for preliminary analysis of the MMETSP resources. This work was supported by The Leverhulme Trust Research project grant RPG-2015-102. T.T., Y.L. and S.M-M. designed the research. T.T. and Y.L. performed analyses. T.T. wrote the manuscript with input from Y.L. and S.M-M. Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Fig. S2 Molecular phylogenetic analysis by the maximum likelihood (ML) method of M1PDH standalones and modules. Fig. S4 Molecular phylogenetic analysis by the maximum likelihood (ML) method of HAD-M1Pase standalones and modules. Fig. S6 Molecular phylogenetic analysis by the maximum likelihood (ML) method of His-M1Pase standalones and module. Table S1 List of algae known to produce mannitol Methods S1 Expanded description of the methods. Notes S5 Expanded version of the Results and Discussion section. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.