Kroth, Peter G.

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Peter G.
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Functional demonstration of Aureochrome 1a proteasomal degradation after blue light incubation in the diatom Phaeodactylum tricornutum

2024, Im, Soo Hyun, Madhuri, Shvaita, Lepetit, Bernard, Kroth, Peter G.

Aureochromes (AUREOs) are both blue light photoreceptors and transcription factors found in diatoms and related algal groups that play a critical role in regulating gene and cell physiology. One of the AUREOs in the diatom Phaeodactylum tricornutum, PtAUREO1a, has been demonstrated to significantly influence global cellular transcription upon blue light exposure. PtAUREO1a itself is highly regulated on the gene transcription level, depending on the light conditions. However, little is known about the proteostasis of PtAUREO1a in vivo. In this study, we used quantitative immunoblot analysis to examine PtAUREO1a levels under different light conditions as well as in the presence of inhibitors for translation and proteolysis. Our results demonstrate that PtAUREO1a is rapidly degraded in response to blue light exposure after red light acclimation, while the protein has an extended protein half-life in white light conditions. Moreover, the data provide the first in vivo evidence for a functional ubiquitin-proteasome system in the model diatom P. tricornutum. Our findings provide a theoretical basis for studies on protein degradation mechanisms and the regulation of PtAUREO1a, suggesting that changing light conditions can have an impact on the PtAUREO1a protein amount by directly affecting its protein stability.

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Identification of promoter targets by Aureochrome 1a in the diatom Phaeodactylum tricornutum

2023, Im, Soo Hyun, Lepetit, Bernard, Mosesso, Niccolò, Shrestha, Sandeep, Weiss, Laura, Nymark, Marianne, Roellig, Robert, Wilhelm, Christian, Isono, Erika, Kroth, Peter G.

Aureochromes (AUREOs) are unique blue light receptors and transcription factors found only in stramenopile algae. While each of the four AUREOs identified in the diatom Phaeodactylum tricornutum may have a specific function, PtAUREO1a has been shown to have a strong impact on overall gene regulation, when light changes from red to blue light conditions. Despite its significance, the molecular mechanism of PtAUREO1a is largely unexplored. To comprehend the overall process of gene regulation by PtAUREO1a, we conducted a series of in vitro and in vivo experiments, including pull-down assays, yeast one-hybrid experiments, and phenotypical characterization using recombinant PtAUREOs and diatom mutant lines expressing a modified PtAureo1a gene. We describe the distinct light absorption properties of four PtAUREOs and the formation of all combinations of their potential dimers. We demonstrate the capability of PtAUREO1a and 1b to activate the genes, diatom-specific cyclin 2, PtAureo1a, and PtAureo1c under both light and dark conditions. Using mutant lines expressing a modified PtAUREO1a protein with a considerably reduced light absorption, we found novel evidence that PtAUREO1a regulates the expression of PtLHCF15, which is essential for red light acclimation. Based on current knowledge, we present a working model of PtAUREO1a gene regulation properties.

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Identification of sequence motifs in Lhcx proteins that confer qE-based photoprotection in the diatom Phaeodactylum tricornutum

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.

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Lhcx proteins provide photoprotection via thermal dissipation of absorbed light in the diatom Phaeodactylum tricornutum

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.

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Prey preference in a kleptoplastic dinoflagellate is linked to photosynthetic performance

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.

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Mitochondrial phosphoenolpyruvate carboxylase contributes to carbon fixation in the diatom Phaeodactylum tricornutum at low inorganic carbon concentrations

2022-08, Yu, Guilan, Nakajima, Kensuke, Gruber, Ansgar, Río Bártulos, Carolina, Schober, Alexander, Lepetit, Bernard, Yohannes, Elizabeth, Matsuda, Yusuke, Kroth, Peter G.

Photosynthetic carbon fixation is often limited by CO2 availability, which led to the evolution of CO2 concentrating mechanisms (CCMs). Some diatoms possess CCMs that employ biochemical fixation of bicarbonate, similar to C4 plants, but it is controversially discussed whether biochemical CCMs are a commonly found in diatoms.
In the diatom Phaeodactylum tricornutum, Phosphoenolpyruvate Carboxylase (PEPC) is present in two isoforms, PEPC1 in the plastids and PEPC2 in the mitochondria. We used real-time quantitative PCR, western blots, and enzymatic assays to examine PEPC expression and PEPC activities, under low and high concentrations of dissolved inorganic carbon (DIC).
We generated and analyzed individual knockout cell lines of PEPC1 and PEPC2, as well as a PEPC1/2 double-knockout strain. While we could not detect an altered phenotype in the PEPC1 knockout strains at ambient, low or high DIC concentrations, PEPC2 and the double-knockout strains grown under ambient air or lower DIC availability, showed reduced growth and photosynthetic affinity to DIC, while behaving similarly as WT cells at high DIC concentrations. These mutants furthermore exhibited significantly lower 13C/12C ratios compared to WT.
Our data implies that in P. tricornutum at least parts of the CCM relies on biochemical bicarbonate fixation catalyzed by the mitochondrial PEPC2.

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The aureochrome photoreceptor PtAUREO1a is a highly effective blue light switch in diatoms

2020-11, Mann, Marcus, Serif, Manuel, Wrobel, Thomas, Eisenhut, Marion, Madhuri, Shvaita, Flachbart, Samantha, Weber, Andreas P. M., Lepetit, Bernard, Wilhelm, Christian, Kroth, Peter G.

Aureochromes represent a unique type of blue-light photoreceptors that possess a blue-light sensing flavin-binding LOV-domain and a DNA-binding bZIP domain, thus being light-driven transcription factors. The diatom Phaeodactylum tricornutum, a member of the essential marine primary producers, possesses four aureochromes (PtAUREO1a, 1b, 1c, 2). We here show a dramatic change in the global gene expression pattern of P. tricornutum wild type cells after a shift from red to blue light. About 75% of the genes show significantly changed transcript levels already after 10 and 60 min of blue light exposure, which includes genes of major transcription factors as well as other photoreceptors. Very surprisingly, in independent PtAureo1a knockout lines, this light-induced regulation of gene expression is almost completely inhibited. Such a massive and fast transcriptional change depending on one single photoreceptor is so far unprecedented. We conclude that PtAUREO1a plays a key role after shifts to blue light in diatoms.

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Effects of dimethoate, an organophosphate insecticide, on photosynthesis of five selected phytoplankton species

2023, Mavrogenis, Martin, Lepetit, Bernard, Kroth, Peter G., Tsirtsis, Georgios

Dimethoate is an organophosphate (OP) insecticide used in agriculture to kill insects. However, information on the effect of the insecticide on non-target organisms like phytoplankton is sparse. When dimethoate enters water ecosystems, it affects the photosynthesis of the phytoplankton cells. The effect of dimethoate on the photosynthesis mechanism of three Chlorophyceaen and two Bacillariophyceaen species was studied measuring oxygen production, chlorophyll fluorescence and xanthophyll pigments. Oxygen evolution rates of the five phytoplankton species decreased with increasing dimethoate concentrations. OJIP fluorescence characteristics in presence of dimethoate was similar to those of DCMU inhibition. DCMU is a PSII inhibitor used as a positive control. Non-photochemical quenching (NPQ) development and the concentration of specific xanthophyll pigments decreased with increasing insecticide concentration, giving another evidence for the target of dimethoate in the selected phytoplankton species. Dark relaxation kinetics did not show a photoinhibition of the cells in presence of dimethoate. Perfekthion, the commercial formulation of dimethoate, inhibited the photosynthesis of the tested phytoplankton species stronger than dimethoate, leading probably to photoinhibition.

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Impact of Lhcx2 on Acclimation to Low Iron Conditions in the Diatom Phaeodactylum tricornutum

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.

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The multifaceted inhibitory effects of an alkylquinolone on the diatom Phaeodactylum tricornutum

2020-04-17, Dow, Lachlan, Stock, Frederike, Peltekis, Alexandra, Szamosvari, David, Prothiwa, Michaela, Lapointe, Adrien, Böttcher, Thomas, Bailleul, Benjamin, Vyverman, Wim, Kroth, Peter G., Lepetit, Bernard

The mechanisms underlying interactions between diatoms and bacteria are crucial to understand diatom behaviour and proliferation, and can result in far-reaching ecological consequences. Recently, 2-alkyl-4-quinolones have been isolated from marine bacteria, both of which (the bacterium and isolated chemical) inhibited growth of microalgae, suggesting these compounds could mediate diatom - bacteria interactions. We investigated the effects of several quinolones on three diatom species. The growth of all three was inhibited, with half-maximal inhibitory concentrations reaching the sub-micromolar range. Using multiple techniques, dual inhibition mechanisms were uncovered for 2-heptyl-4-quinolone (HHQ) in Phaeodactylum tricornutum. Firstly, photosynthetic electron transport was obstructed, primarily via inhibition of the cytochrome b6f complex. Secondly, respiration was inhibited, leading to repression of ATP supply to plastids from mitochondria via organelle energy coupling. These data clearly show how HHQ could modulate diatom proliferation in marine environments.