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. 2022 Apr 11;39(4):msac065.
doi: 10.1093/molbev/msac065.

An Enigmatic Stramenopile Sheds Light on Early Evolution in Ochrophyta Plastid Organellogenesis

Tomonori Azuma  1 , Tomáš Pánek  2   3 , Alexander K Tice  3 , Motoki Kayama  1 , Mayumi Kobayashi  4 , Hideaki Miyashita  1 , Toshinobu Suzaki  4 , Akinori Yabuki  5 , Matthew W Brown  3 , Ryoma Kamikawa  6
Affiliations

An Enigmatic Stramenopile Sheds Light on Early Evolution in Ochrophyta Plastid Organellogenesis

Tomonori Azuma et al. Mol Biol Evol. .

Abstract

Ochrophyta is an algal group belonging to the Stramenopiles and comprises diverse lineages of algae which contribute significantly to the oceanic ecosystems as primary producers. However, early evolution of the plastid organelle in Ochrophyta is not fully understood. In this study, we provide a well-supported tree of the Stramenopiles inferred by the large-scale phylogenomic analysis that unveils the eukaryvorous (nonphotosynthetic) protist Actinophrys sol (Actinophryidae) is closely related to Ochrophyta. We used genomic and transcriptomic data generated from A. sol to detect molecular traits of its plastid and we found no evidence of plastid genome and plastid-mediated biosynthesis, consistent with previous ultrastructural studies that did not identify any plastids in Actinophryidae. Moreover, our phylogenetic analyses of particular biosynthetic pathways provide no evidence of a current and past plastid in A. sol. However, we found more than a dozen organellar aminoacyl-tRNA synthases (aaRSs) that are of algal origin. Close relationships between aaRS from A. sol and their ochrophyte homologs document gene transfer of algal genes that happened before the divergence of Actinophryidae and Ochrophyta lineages. We further showed experimentally that organellar aaRSs of A. sol are targeted exclusively to mitochondria, although organellar aaRSs in Ochrophyta are dually targeted to mitochondria and plastids. Together, our findings suggested that the last common ancestor of Actinophryidae and Ochrophyta had not yet completed the establishment of host-plastid partnership as seen in the current Ochrophyta species, but acquired at least certain nuclear-encoded genes for the plastid functions.

Keywords: Actinophryidae; aminoacyl-tRNA synthase; gene transfer; organellar DNA; phylogenomics; plastid evolution.

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Figures

Fig. 1.
Fig. 1.
Phylogenomic tree of the Stramenopiles. Tree reconstruction was conducted by ML analysis with a dataset comprised of 75 taxa and 75,984 amino acid sites under LG + G4 + F + C60-PMSF model. Species of Alveolata and Rhizaria are regarded as outgroup taxa. The numbers on branches are the ML bootstrap values. If not shown, the nodes are fully supported.
Fig. 2.
Fig. 2.
Representative metabolisms deduced from the transcriptome data of Actinophrys sol. Whereas gray lines and circles show undetected biochemical reactions and involved proteins, respectively, colored lines show those deduced from the transcriptome data. Light green circles enclosed by green lines show proteins with detectable mitochondria-targeting sequences. Light pink circles enclosed by pink lines show possible ER proteins with detectable signal peptides but lacking transit peptide-like regions and the ASAFAP motif. Light blue circles enclosed by purple lines show cytosolic proteins. Circles lacking enclosing lines show proteins that are N-terminally truncated. AACT, acetoacetyl-CoA thiolase; AaRS, aminoacyl-tRNA synthase; ACC, acetyl-CoA carboxylase; ACO, aconitate hydratase; ACP, acyl carrier protein; BOLA1, ISC targeting factor; BOLA3, ISC targeting factor; CDP-DAGS, CDP-diacylglycerol synthase; CFD1, iron–sulfur cluster assembly protein Cfd1CIA1, CIA targeting complex Cia1; CIA2, CIA targeting complex Cia2; CS, citrate synthase; DGAT, diacylglycerol acyltransferase; DGK, diacylglycerol kinase; DHST, dihydrolipoamide succinyltransferase; DRE2, electron carrier Dre2; ECH, enoyl-CoA hydratase; EL, enolase; FabD, malonyl-CoA:ACP transacylase; FabF, 3-oxoacyl-ACP synthase II; FabG, 3-oxoacyl-ACP reductase; FabI, enoyl-ACP reductase; FabZ, 3-hydroxyacyl-ACP dehydratase; FBA, fructose 1,6-bisphosphate aldolase Class I; FBP, fructose-1,6-bisphosphatase; FDX2, ferredoxin; FDXR, ferredoxin reductase; FH, fumarate hydratase, Class I; FXN, frataxin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GCSH, glycine cleavage system H protein; GCSL, glycine cleavage system L protein; GCSP, glycine cleavage system P protein; GCST, glycine cleavage system T protein; GK, glucokinase; GLRX5, monothiol glutaredoxin; GPAT, glycerol-3-phosphate O-acyltransferase; GPDH, glucose-6-phosphate 1-dehydrogenase; GPI, glucose-6-phosphate isomerase; GPM, phosphoglucomutase; GRPE1, nucleotide exchange factor; HAD, 3-hydroxyacyl-CoA dehydrogenase; HMGR, hydroxymethylglutaryl-CoA reductase; HMGS, hydroxymethylglutaryl-CoA synthase; HSC20, J-type cochaperone; HSPA9, Hsp70 chaperone; IBA57, ISC protein; IDH, isocitrate dehydrogenase; IND1, [4Fe–4S] cluster-binding protein; ISCA1, ISC protein; ISCA2, ISC protein; ISCU2, scaffold protein; ISD11, cysteine desulfurase; KT, 3-ketoacyl-CoA thiolase; LCAD, long-chain-acyl-CoA dehydrogenase; LPAT, lysophosphatidate acyltransferase; MCAD, medium-chain-acyl-CoA dehydrogenase; MDC, diphosphomevalonate decarboxylase; MDH, malate dehydrogenase; MMS19, CIA targeting complex Mms19; MVK, mevalonate kinase; NAR1, iron–sulfur cluster assembly protein Nar1NBP35, CIA scaffold protein Nbp35; NFS1, cysteine desulfurase; NFU1, [4Fe–4S] cluster-binding protein; OGDH, 2-oxoglutarate dehydrogenase E1 component; PAP, phosphatidic acid phosphatase; PDAT, phospholipid:diacylglycerol acyltransferase; PDH E1a, pyruvate dehydrogenase E1 subunit alpha protein; PDH E1b, pyruvate dehydrogenase E1 subunit beta protein; PDH E2, pyruvate dehydrogenase E2 subunit; PDH E3, pyruvate dehydrogenase E3 subunit; PFK, phosphofructokinase; PGDH, 6-phosphogluconate dehydrogenase; PGK, phosphoglycerate kinase; PGL, 6-phosphogluconolactonase; PGM, phosphoglycerate mutase; PGPP, phosphatidylglycerophosphatase; PGPS, phosphatidylglycerophosphate synthase; PK, pyruvate kinase; PMK, phosphomevalonate kinase; POP, plant and protist organellar DNA polymerase; RP, ribosomal protein; RPE, ribulose-phosphate 3-epimerase; RPO, RNA polymerase; SCAD, short-chain-acyl-CoA dehydrogenase; Sec61, Sec61 complex; SELMA, symbiont-specific ER-associated degradation (ERAD)-like machinery; SDH, succinate dehydrogenase; SUCLG1, succinyl-CoA synthetase alpha subunit; SUCLG2, succinyl-CoA synthetase beta subunit; TAH18, diflavin reductase Tah18; TAL, transaldolase; TIC/TOC, translocon at the inner/outer envelope membrane of chloroplasts; TKL, transketolase; TPI, triose phosphate isomerase; TPT, triose phosphate transporter.
Fig. 3.
Fig. 3.
Evolution and localization of organellar aminoacyl-tRNA synthases in A. sol. (A) ML tree of GluRS. The “plastid clade” comprising plastid-bearing species and A. sol is highlighted. The numbers on branches represent bootstrap values. Only bootstrap values ≥50 are shown. Clades outside of our interest are collapsed as closed triangles and the numbers of taxa are indicated. Prokaryotic sequences are in light gray. Note that Paraphysomonas possesses a nonphotosynthetic plastid that does not contain DNA and its aaRSs of the PL-clade target to the mitochondria (Dorrell et al. 2019). (B–D) ML trees for LeuRS, ProRS, and ThrRS. Cafeteria roenbergensis, Opalozoa, is highlighted in orange in (C). Other details are as same as (A). (E) N-terminal amino acid sequences of A. sol aaRSs, C-terminally tagged by GFPs. The underlined regions are mitochondrial transit peptides predicted by the in silico analyses. (F) Localization of the recombinant GFPs. Arrowheads indicate compartments, in which the GFP recombinant proteins localize. GFP fluorescence is not colocalized with chlorophyll fluorescence in “Mitotracker (−)”, but colocalized with Mitotracker fluorescence in “Mitotracker (+)”, indicative of mitochondrial localization. DIC, differential interference contrast; Chl, chlorophyll autofluorescence; GFP, GFP fluorescence; Merge, a merged image of chlorophyll and GFP fluorescence. Scale bars in the pictures show 10 µm.
Fig. 4.
Fig. 4.
Possible scenarios for early plastid evolution in Ochrophyta. (A) Detected plastid-related genes (e.g., aaRS) found in A. sol are derived from independent lateral gene transfers not associated with endosymbiosis of a photosynthetic eukaryote. After divergence from Actinophryidae, an ancestor of Ochrophyta has obtained a plastid. (B) Plastid-related genes (e.g., aaRS) found in A. sol are derived from endosymbiotic gene transfers occurred in the common ancestor of Actinophryidae and Ochrophyta. The endosymbiont gave rise to the fully integrated ochrophyte plastid. In contrast, the ancestor of Actinophryidae has lost the endosymbiont. (C) This is the similar scenario with (B). However, the endosymbiont has been replaced by a new one that gave rise to current ochrophyte plastid. (D) The common ancestor of Actinophryidae and Ochrophyta has possessed a plastid, followed by loss of a plastid in the evolution of Actinophryidae. However, the host cell in the common ancestor of Actinophryidae and Ochrophyta has retained both host-derived genuine genes and metabolic pathways in addition to their corresponding plastid-derived metabolic pathways and genes.
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