2023-06-30, Yamada, Norico, Lepetit, Bernard, Mann, David G., Sprecher, Brittany N., Buck, Jochen Mario, Bergmann, Paavo, Kroth, Peter G., Bolton, John J., Dąbek, Przemysław, Witkowski, Andrzej

Dinoflagellates of the family Kryptoperidiniaceae, known as “dinotoms”, possess diatom-derived endosymbionts and contain individuals at three successive evolutionary stages: a transiently maintained kleptoplastic stage; a stage containing multiple permanently maintained diatom endosymbionts; and a further permanent stage containing a single diatom endosymbiont. Kleptoplastic dinotoms were discovered only recently, in Durinskia capensis ; until now it has not been investigated kleptoplastic behavior and the metabolic and genetic integration of host and prey. Here, we show D. capensis is able to use various diatom species as kleptoplastids and exhibits different photosynthetic capacities depending on the diatom species. This is in contrast with the prey diatoms in their free-living stage, as there are no differences in their photosynthetic capacities. Complete photosynthesis including both the light reactions and the Calvin cycle remain active only when D. capensis feeds on its habitual associate, the “essential” diatom Nitzschia captiva . The organelles of another edible diatom, N. inconspicua , are preserved intact after ingestion by D. capensis and expresses the psbC gene of the photosynthetic light reaction, while RuBisCO gene expression is lost. Our results indicate that edible but non-essential, “supplemental” diatoms are used by D. capensis for producing ATP and NADPH, but not for carbon fixation. D. capensis has established a species-specifically designed metabolic system allowing carbon fixation to be performed only by its essential diatoms. The ability of D. capensis to ingest supplemental diatoms as kleptoplastids may be a flexible ecological strategy, to use these diatoms as “emergency supplies” while no essential diatoms are available.

2021-12, Buck, Jochen Mario, Kroth, Peter G., Lepetit, Bernard

Photosynthetic organisms in nature often experience light fluctuations. While low light conditions limit the energy uptake by algae, light absorption exceeding the maximal rate of photosynthesis may go along with enhanced formation of potentially toxic reactive oxygen species. To preempt high light induced photodamage, photosynthetic organisms evolved numerous photoprotective mechanisms. Amongst these, energy-dependent fluorescence quenching (qE) provides a rapid mechanism to thermally dissipate excessively absorbed energy.

Diatoms thrive in all aquatic environments and thus belong to the most important primary producers on earth. qE in diatoms is provided by a concerted action of Lhcx proteins and the xanthophyll cycle pigment diatoxanthin. While the exact Lhcx activation mechanism of diatom qE is unknown, two lumen-exposed acidic amino acids within Lhcx proteins were proposed to function as regulatory switches upon light induced lumenal acidification. By introducing a modified Lhcx1 lacking these amino acids into a Phaeodactylum tricornutum Lhcx1-null qE knockout line, we demonstrate that qE is unaffected by these two amino acids. Based on sequence comparisons with Lhcx4, being incapable of providing qE, we perform domain swap experiments of Lhcx4 with Lhcx1 and identify two peptide motifs involved in conferring qE. Within one of these motifs, we identify a tryptophan residue with a major influence on qE establishment. This tryptophan residue is located in close proximity to the diadinoxanthin/diatoxanthin binding site based on the recently revealed diatom Lhc crystal structure. Our findings provide a structural explanation for the intimate link of Lhcx and diatoxanthin in providing qE in diatoms.

2023-05, Sprecher, Brittany N., Buck, Jochen Mario, Ropella, L. Loraine, Ramsperger, Annette, Kroth, Peter G., Yamada, Norico

Endosymbiosis is a widespread and ecologically significant phenomenon in the marine environment. How these endosymbiotic partners evolve into an organism with a new organelle is still mostly unknown and requires investigation into modern symbioses. Dinotoms, dinoflagellates with evolutionarily intermediate diatom plastids, are considered excellent models for studying organellogenesis as they remain at three successive but distinct stages. Efforts to understand the host dinoflagellate-endosymbiotic diatom relationship has been limited by the lack of genetic transformation methods for either member of the symbiosis. To address this absence, we modified existing diatom biolistic and conjugation transformation methods and cryopreservation protocols for the diatom Nitzschia captiva, an essential prey for the kleptoplastic dinotom Durinskia capensis. Through the use of Phaeodactylum tricornutum, Cylindrotheca fusiformis, and native Nitzschia captiva diatom designed plasmids, we successfully express and target EGFP to the cytosol, mitochondria, and plastids of N. captiva, and visualize these organelles inside D. capensis in vivo, allowing specific labeling and tracking of organelles and proteins after ingestion. Furthermore, we attempt to utilize CRISPR/Cas9 to target the introduced EGFP gene but find no evidence of successful gene editing.

2019-09-13, Buck, Jochen Mario, Sherman, Jonathan, Río Bártulos, Carolina, Serif, Manuel, Halder, Marc, Henkel, Jan, Falciatore, Angela, Lavaud, Johann, Kroth, Peter G., Lepetit, Bernard

Diatoms possess an impressive capacity for rapidly inducible thermal dissipation of excess absorbed energy (qE), provided by the xanthophyll diatoxanthin and Lhcx proteins. By knocking out the Lhcx1 and Lhcx2 genes individually in Phaeodactylum tricornutum strain 4 and complementing the knockout lines with different Lhcx proteins, multiple mutants with varying qE capacities are obtained, ranging from zero to high values. We demonstrate that qE is entirely dependent on the concerted action of diatoxanthin and Lhcx proteins, with Lhcx1, Lhcx2 and Lhcx3 having similar functions. Moreover, we establish a clear link between Lhcx1/2/3 mediated inducible thermal energy dissipation and a reduction in the functional absorption cross-section of photosystem II. This regulation of the functional absorption cross-section can be tuned by altered Lhcx protein expression in response to environmental conditions. Our results provide a holistic understanding of the rapidly inducible thermal energy dissipation process and its mechanistic implications in diatoms.

2022-03-16, Buck, Jochen Mario, Wünsch, Marie, Schober, Alexander, Kroth, Peter G., Lepetit, Bernard

Iron is a cofactor of photosystems and electron carriers in the photosynthetic electron transport chain. Low concentrations of dissolved iron are, therefore, the predominant factor that limits the growth of phototrophs in large parts of the open sea like the Southern Ocean and the North Pacific, resulting in “high nutrient–low chlorophyll” (HNLC) areas. Diatoms are among the most abundant microalgae in HNLC zones. Besides efficient iron uptake mechanisms, efficient photoprotection might be one of the key traits enabling them to outcompete other algae in HNLC regions. In diatoms, Lhcx proteins play a crucial role in one of the main photoprotective mechanisms, the energy-dependent fluorescence quenching (qE). The expression of Lhcx proteins is strongly influenced by various environmental triggers. We show that Lhcx2 responds specifically and in a very sensitive manner to iron limitation in the diatom Phaeodactylum tricornutum on the same timescale as the known iron-regulated genes ISIP1 and CCHH11. By comparing Lhcx2 knockout lines with wild type cells, we reveal that a strongly increased qE under iron limitation is based on the upregulation of Lhcx2. Other observed iron acclimation phenotypes in P. tricornutum include a massively reduced chlorophyll a content/cell, a changed ratio of light harvesting and photoprotective pigments per chlorophyll a, a decreased amount of photosystem II and photosystem I cores, an increased functional photosystem II absorption cross section, and decoupled antenna complexes. H2O2 formation at photosystem I induced by high light is lowered in iron-limited cells, while the amount of total reactive oxygen species is rather increased. Our data indicate a possible reduction in singlet oxygen by Lhcx2-based qE, while the other iron acclimation phenotype parameters monitored are not affected by the amount of Lhcx2 and qE.

2018, Buck, Jochen Mario, Río Bártulos, Carolina, Gruber, Ansgar, Kroth, Peter G.

Most genetic transformation protocols for the model diatom Phaeodactylum tricornutum rely on one of two available antibiotics as selection markers: Zeocin (a formulation of phleomycin D1) or nourseothricin. This limits the number of possible consecutive genetic transformations that can be performed. In order to expand the biotechnological possibilities for P. tricornutum, we searched for additional antibiotics and corresponding resistance genes that might be suitable for use with this diatom. Among the three different antibiotics tested in this study, blasticidin-S and tunicamycin turned out to be lethal to wild-type cells at low concentrations, while voriconazole had no detectable effect on P. tricornutum. Testing the respective resistance genes, we found that the blasticidin-S deaminase gene (bsr) effectively conferred resistance against blasticidin-S to P. tricornutum. Furthermore, we could show that expression of bsr did not lead to cross-resistances against Zeocin or nourseothricin, and that genetically transformed cell lines with resistance against Zeocin or nourseothricin were not resistant against blasticidin-S. In a proof of concept, we also successfully generated double resistant (against blasticidin-S and nourseothricin) P. tricornutum cell lines by co-delivering the bsr vector with a vector conferring nourseothricin resistance to wild-type cells.