Light regimes are a fundamental environmental cue for marine organisms, yet molecular adaptations of photoreceptors to the extreme seasonality of polar regions remain poorly understood. We demonstrate that the diversity of candidate blue-light-sensing LOV-domain proteins increases towards higher latitudes. We identify Aureochromes, blue-light-sensitive transcription factors unique to stramenopiles, as the most frequent LOV-domain receptors across ocean latitudes. Phylogenetic reconstructions reveal that Aureochromes diversified early in the stramenopile lineage, originating in a heterotrophic ancestor before the acquisition of photosynthesis. This challenges the assumption that Aureochromes are restricted to phototrophs, suggesting an ancestral role in spectral light signaling. Despite strong latitudinal shifts in light quality and photoperiod, diatom Aureochrome domain architecture remains conserved, implying adaptation through regulatory tuning (e.g. altered expression dynamics or post-translational control). Together, our results emphasise an importance of spectral-light sensing in polar oceans and link the widespread dominance of Aureochromes to their ancient origin and conservation.

Phasing biological and physiological processes to periodic light-dark cycles is crucial for the life of most organisms. Marine diatoms, as many phytoplanktonic species, exhibit biological rhythms, yet their molecular timekeepers remain largely uncharacterized. Recently, the bHLH-PAS protein RITMO1 has been proposed to act as a regulator of diatom circadian rhythms. In this study, we first determined the physiological conditions to monitor circadian clock activity and its perturbation in the diatom model species Phaeodactylum tricornutum by using cell fluorescence as a circadian output. Employing ectopic overexpression, targeted gene mutagenesis, and functional complementation, we then investigated the role of RITMO1 in various circadian processes. Our data reveal that RITMO1 significantly influences the P. tricornutum circadian rhythms not only of cellular fluorescence, but also of photosynthesis and of the expression of clock-controlled genes, including transcription factors and putative clock input/output components. RITMO1 effects on rhythmicity are unambiguously detectable under free-running conditions. By uncovering the complex regulation of biological rhythms in P. tricornutum, these findings advance our understanding of the endogenous factors controlling diatom physiological responses to environmental changes. They also offer initial insights into the mechanistic principles of oscillator functions in a major group of phytoplankton, which remain largely unexplored in chronobiology.

Diatoms dominate phytoplankton communities in turbulent waters, where light fluctuations can be frequent and intense. Due to this complex environment, these heterokont microalgae display remarkable photoprotection strategies, including a fast Non-Photochemical Quenching (NPQ). However, in nature, several abiotic parameters (such as temperature) can influence the response of photosynthetic organisms to light stress in a synergistic or antagonistic manner. Yet, the combined effects of light and these other drivers on the photosynthetic and photoprotective capacity of diatoms are still poorly understood. In this work, we investigated the impact of short-term temperature and light stress on the model diatom Phaeodactylum tricornutum, combining NPQ induction-recovery assays or light curves with a broad gradient of superimposed temperature treatments (5 to 35°C). We employed mutant lines deficient in NPQ generation (vde KO) or recovery (zep3 KO) and wild type. We found that temperature and light have a synergistic effect: lower temperatures limited both the photosynthetic capacity and NPQ, while the general photophysiological performance was enhanced with warming, up to a heat-stress limit (above 30°C). We discuss the temperature effects on NPQ induction and recovery and propose that these are independent from the energy requirements of the cells and result from altered xanthophyll cycle dynamics. Namely, we found that de-epoxidation activity strongly increases with temperature, outweighing epoxidation and resulting in a positive increase of NPQ with temperature. Finally, we propose that in a short-term time frame, temperature and light have a synergistic and not antagonistic effect, with a positive relationship between increasing temperature and NPQ.

Tertiary plastids derived from diatoms in "dinotom" dinoflagellates offer a rare view of organellogenesis in action, while the genomic and metabolic processes underlying their conversion remain poorly understood. Here, we present a comparative transcriptomic analysis of two dinotoms at different plastidial levels: Durinskia capensis at the kleptoplastidy state, alongside its kleptoplastid-source diatom Nitzschia captiva, and its close relative Durinskia kwazulunatalensis at an early permanent state. We show that in both dinotoms, the diatom nucleus retains high transcriptional autonomy, but its expression profile is plastid biased, signaling early host influence. In contrast, only D. kwazulunatalensis exhibits striking signs of genomic reconfiguration in the diatom nucleus: intron insertions, increased guanine (G) and cytosine (C) content, and growing nucleotide similarity to host transcripts. These shifts suggest an incipient nucleomorph-like transformation. Metabolically, only D. kwazulunatalensis expresses a complete hexose phosphate export pathway, suggesting deeper metabolic integration, while both species retain simpler carbohydrate transport routes. Additionally, we propose that diatom karyokinesis might be controlled by a dual mechanism via suppression of key transcription factors at the G1-S checkpoint and nitrate availability. Together, our findings reveal a continuum of plastid integration degrees, from temporary organelle theft to genomic accommodation and metabolic codependence. Dinotoms thus serve not only as evolutionary artifacts but as living laboratories, illuminating how kleptoplastids inch toward permanence.

Xanthophyll cycling contributes to photoprotection by regulating nonphotochemical quenching (NPQ), a form of excess energy dissipation through heat. While most photosynthetic eukaryotes (including land plants) use the violaxanthin cycle, some algae like diatoms and haptophytes rely on the diadinoxanthin cycle for photoprotection. These algae also contain minor amounts of violaxanthin cycle pigments, thought to serve only as the precursors of other major xanthophylls. Both cycles are catalyzed by the enzymes violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP). Here, we characterized the role of VDE and of the ZEP paralogs ZEP2 and ZEP3 in the model diatom Phaeodactylum tricornutum. We generated knockout lines for each gene and treated exponentially growing mutants and wild type with periodic high-light stress. As knockouts of VDE and ZEP3 were significantly impaired in the diadinoxanthin cycle, we concluded that VDE and ZEP3 are the main regulators of the diadinoxanthin cycle in this diatom. Strikingly, under light stress, ZEP2 knockouts mainly accumulated pigments of the violaxanthin cycle instead of the diadinoxanthin cycle, but still displayed the same NPQ capacity as the wild type. We conclude that both major xanthophyll cycles present in nature can contribute to NPQ with comparable efficiency within the same diatom species, offering perspective on the evolution of xanthophyll-mediated photoprotection.

Achnanthidium minutissimum is a widely distributed benthic freshwater diatom. The alga can produce stalks that stick the cell to the surface and subsequently extracellular capsules developing into biofilms. Extracts of the diatom-associated bacterium Dyadobacter sp. 32 have been shown previously to induce stalk and capsule formation by the diatom. Here, we studied the impact of macronutrients on the generation of stalks induced by bacterial extracts with respect to the frequency of stalk generation and stalk lengths, using axenic cultures to avoid any additional impact of bacteria on the nutrient availability. We found that nitrate deprivation inhibited cell division of A. minutissimum within four days, but it did not initially affect stalk production or elongation. Silica limitation instead inhibited both stalk production and elongation. Similarly, sulfate was required for stalk formation, which was supported by the energy-dispersive X-ray spectroscopy of A. minutissimum cells showing that sulfur was abundant in the stalks.

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.

Plants and algae use light not only for driving photosynthesis but also to sense environmental cues and to adjust their circadian clocks via photoreceptors. Aureochromes are blue-light-dependent photoreceptors that also function as transcription factors, possessing both a LOV and a bZIP domain. Aureochromes so far have only been detected in Stramenopile algae, which include the diatoms. Four paralogues of aureochromes have been identified in the pennate model diatom Phaeodactylum tricornutum: PtAureo1a, 1b, 1c, and 2. While it was shown recently that diatoms have a diel rhythm, the molecular mechanisms and components regulating it are still largely unknown. Diel gene expression analyses of wild-type P. tricornutum, a PtAureo1a knockout strain, and the respective PtAureo1 complemented line revealed that all four aureochromes have a different diel regulation and that PtAureo1a has a strong co-regulatory influence on its own transcription, as well as on that of other genes encoding different blue-light photoreceptors (CPF1, 2 and 4), proteins involved in photoprotection (Lhcx1), and specific bHLH transcription factors (RITMO1). Some of these genes completely lost their circadian expression in the PtAureo1a KO mutant. Our results suggest a major involvement of aureochromes in the molecular clock of diatoms.

Polyphosphates (polyP) are ubiquitous biomolecules that play a multitude of physiological roles in many cells. We have studied the presence and role of polyP in a unicellular alga, the freshwater diatom Achnanthidium minutissimum. This diatom stores up to 2.0 pg·cell-1 of polyP, with chain lengths ranging from 130 to 500 inorganic phosphate units (Pi). We applied energy dispersive X-ray spectroscopy, Raman/fluorescence microscopy, and biochemical assays to localize and characterize the intracellular polyP granules that were present in large apical vacuoles. We investigated the fate of polyP in axenic A. minutissimum cells grown under phosphorus (P), replete (P(+)), or P deplete (P(-)) cultivation conditions and observed that in the absence of exogenous P, A. minutissimum rapidly utilizes their internal polyP reserves, maintaining their intrinsic growth rates for up to 8 days. PolyP-depleted A. minutissimum cells rapidly took up exogenous P a few hours after Pi resupply and generated polyP three times faster than cells that were not initially subjected to P limitation. Accordingly, we propose that A. minutissimum deploys a succession of acclimation strategies regarding polyP dynamics where the production or consumption of polyP plays a central role in the homeostasis of the diatom.

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.

Photosynthetic organisms require light but also rely on photoprotection to preempt photodamage induced by excess light. The xanthophyll cycle, a light-dependent carotenoid interconversion, plays a key role in the on- and offset of Non-Photochemical Quenching (NPQ), a form of excess energy dissipation through heat. While in most photosynthetic eukaryotes, including brown algae, green algae and plants, the violaxanthin cycle is prevalent, haptophytes and diatoms rely on the diadinoxanthin cycle to regulate NPQ. Diatoms also contain small amounts of violaxanthin cycle pigments, thought to serve only as precursors in xanthophylls biosynthesis. Both cycles are catalyzed by the enzymes violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP). Here, we characterized the role of VDE and different ZEP encoding paralogs ( ZEP2 and ZEP3 ) in the model diatom Phaeodactylum tricornutum . We generated the respective knockout lines and treated exponentially growing mutants and wild type with periodic high light stress. We conclude that VDE and ZEP3 are the main regulators of the diadinoxanthin cycle. Under the same conditions, ZEP2 knockouts accumulated mainly pigments of the violaxanthin cycle instead of the diadinoxanthin cycle. Intriguingly, violaxanthin cycle pigments contributed to the generation of NPQ with the same quenching efficiency of diadinoxanthin cycle pigments, demonstrating that both major xanthophyll cycles present in nature can provide NPQ in the same organism, with similar properties. Consequently, the prevalence of the diadinoxanthin cycle in diatoms has not been driven by its higher quenching efficiency but likely resulted from the selective advantage of a faster switch between photoprotection and light harvesting.Significance Statement Diatoms have a key role in almost any aquatic habitat, participate in nutrient cycling and contribute as much as the most productive terrestrial ecosystems to the global primary productivity. Diatoms are also regarded as biological factories of high value bioactive compounds, like carotenoids. Here, we investigated one of the most significant photoprotection mechanisms, the xanthophyll cycle, which relies on carotenoids to dissipate detrimental excess of light energy. Although diatoms use the so-called diadinoxanthin cycle, we discovered that the ancestral violaxanthin cycle, ubiquitous in plants and algae, can also contribute to photoprotection in these algae. We demonstrate that both cycles can function synergistically and with comparable efficiency within the same species, offering a new perspective on the evolution of xanthophyll-mediated photoprotection.### Competing Interest StatementThe authors have declared no competing interest.

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.

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.

The author declares that there is no conflict of interest.

Summary 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 whether biochemical CCMs are commonly found in diatoms is a subject of debate. 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 polymerase chain reaction, Western blots, and enzymatic assays to examine PEPC expression and PEPC activity, 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 conditions showed reduced growth and photosynthetic affinity for DIC while behaving similarly to wild‐type (WT) cells at high DIC concentrations. These mutants furthermore exhibited significantly lower 13C/12C ratios compared to the WT. Our data imply that in P. tricornutum at least parts of the CCM rely on biochemical bicarbonate fixation catalyzed by the mitochondrial PEPC2.

Diatoms are phototrophic, unicellular, and eukaryotic organisms. They originate from secondary endosymbiosis, a specific evolutionary process. Accordingly, their cells and organelles have a typical organisation, as revealed by ultrastructural investigations. Diatoms possess specific compartments and structures, including a silica shell surrounding the diatom cell, the so-called silica deposition vesicles (SDVs), as well as complex plastids that are surrounded by four membranes. Here we provide an overview of diatom organelles, and recapitulate recent information obtained from 3D imaging of whole diatom cells, focusing on the subcellular topology of the model diatom Phaeodactylum tricornutum. This chapter will not discuss issues of the cell wall and the SDVs, which are covered in Chaps. “Structure and Morphogenesis of the Frustule” and “Biomolecules Involved in Frustule Biogenesis and Function”.

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.

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. Among these, energy-dependent fluorescence quenching (qE) provides a rapid mechanism to dissipate thermally the 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.