Kroth, Peter G.
0000-0003-4734-8955 Berufsbeschreibung
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Mitochondrial phosphoenolpyruvate carboxylase contributes to carbon fixation in the diatom Phaeodactylum tricornutum at low inorganic carbon concentrations
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.
Cell Biology of Organelles
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”.
Identification of sequence motifs in Lhcx proteins that confer qE-based photoprotection in the diatom Phaeodactylum tricornutum
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.
Complete genome sequence of Dyadobacter sp. 32, isolated from a culture of the freshwater diatom Cymbella microcephala
Bacteria have been shown to be involved in different species-specific interactions with eukaryotic algae such as diatoms, impacting important ecosystem processes. Recently, a strain assigned to Dyadobacter, named ‘species 32’, has been shown to be involved in a number of ecologically relevant diatom processes, such as biofilm formation or growth enhancement, depending on the diatom species. This bacterium was originally isolated from a culture of freshwater benthic diatoms that originated from an epilithic biofilm, in which both bacteria and diatoms coexist. A single complete circular chromosome of Dyadobacter sp. 32 was assembled with a length of 7,101,228 bp, containing 6062 protein coding genes and 3 rRNA operons. A number of interesting genetic features were found, such as a putative zeaxanthin biosynthetic gene cluster. A large number of polysaccharide utilizing gene clusters were also detected, along with genes potentially acquired from other bacteria through horizontal gene transfer, and genes previously identified in other algae-bacteria interactions. These data serve to increase our understanding of specific interactions within freshwater biofilms, and identify a number of gene targets with which to study the molecular basis of diatom-bacteria interactions.
Impact of Lhcx2 on Acclimation to Low Iron Conditions in the Diatom Phaeodactylum tricornutum
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.
Carbohydrate Metabolism
Diatoms are unicellular algae that perform photosynthesis, a process that comprises both the electron transport processes in the thylakoid membranes and the reactions involved in CO2 fixation. The latter process is the starting point for carbohydrate biosynthesis and numerous reactions and pathways in different cellular locations, including the generation, modification, conversion, subsequent storage, and degradation of carbohydrates. While there is vast knowledge of these processes available of land plants and green algae, much less is known of algae like diatoms that are derived from secondary endosymbiosis. Comparative studies on the localization and regulation of photosynthetic pathways in recent years revealed in principle a similarity of photosynthesis in land plants and diatoms, but also a number of peculiar differences, which may be due both to the general phylogenetic distance between these groups and the evolution of diatoms by secondary endosymbiosis, resulting in a different genetic background. This chapter describes the current knowledge of CO2 acquisition and fixation processes in diatoms on the molecular, cellular, and physiological levels in diatoms. Additional focus is laid on photorespiration, as well as carbohydrate pathways, carbohydrate degradation, and carbohydrate storage.
Five Non-motile Dinotom Dinoflagellates of the Genus Dinothrix
Dinothrix paradoxa and Gymnodinium quadrilobatum are benthic dinoflagellates possessing diatom-derived tertiary plastids, so-called dinotoms. Due to the lack of available genetic information, their phylogenetic relationship remains unknown. In this study, sequencing of 18S ribosomal DNA (rDNA) and the rbcL gene from temporary cultures isolated from natural samples revealed that they are close relatives of another dinotom, Galeidinium rugatum. The morphologies of these three dinotoms differ significantly from each other; however, they share a distinctive life cycle, in which the non-motile cells without flagella are their dominant phase. Cell division occurs in this non-motile phase, while swimming cells only appear for several hours after being released from each daughter cell. Furthermore, we succeeded in isolating and establishing two novel dinotom strains, HG180 and HG204, which show a similar life cycle and are phylogenetically closely related to the aforementioned three species. The non-motile cells of strain HG180 are characterized by the possession of a hemispheroidal cell covered with numerous nodes, while those of the strain HG204 form aggregations consisting of spherical smooth-surface cells. Based on the similarity in life cycles and phylogenetic closeness, we conclude that all five species should belong to a single genus, Dinothrix, the oldest genus within this clade. We transferred Ga. rugatum and Gy. quadrilobatum to Dinothrix, and described strains HG180 and HG204 as Dinothrix phymatodea sp. nov. and Dinothrix pseudoparadoxa sp. nov.
High throughput method for extracting polyphosphates from diatoms
Polyphosphates (polyP) are widely distributed in living organisms and have crucial cellular functions. Little is known about the physiological roles of polyP in algae, especially in diatoms. Therefore, we have developed a standardized method for polyP extraction and quantification from diatoms, using the freshwater model organism A. minutissimum and further marine diatoms to validate the method. Investigating the efficiency of polyP extraction methods, we found that a commercial DNA isolation kit yielded best results. The method is based on separation of polyP from lower molecular weight compounds using gel filtration spin columns. Moreover, we demonstrate that self-made gel filtration spin columns and extraction buffers can also extract polyP from A. minutissimum efficiently. Under defined conditions, samples spiked with polyP of chain-lengths between 45 to 700 Pi moieties consistently resulted in a recovery of more than 70% of the initially loaded polyP. PolyP was quantified with an ascorbate-antimony-molybdate assay that detects phosphates (Pi) by hydrolysis of a specific exopolyphosphatase (PPX). The method detects as little as 3 μM polyP in high throughput analyses using 96-well plates. This sensitive, rapid and straightforward method is ideal to characterize the physiological role of polyP in diatoms.
Sensing and Signalling in Diatom Responses to Abiotic Cues
Diatoms are prominent microalgae that proliferate in a wide range of aquatic environments. Still, fundamental questions regarding their biology, such as how diatoms sense and respond to environmental variations, remain largely unanswered. In recent years, advances in the molecular and cell biology of diatoms and the increasing availability of genomic data have made it possible to explore sensing and signalling pathways in these algae. Pivotal studies of photosensory perception have highlighted the great capacity of diatoms to accurately detect environmental variations by sensing differential light signals and adjust their physiology accordingly. The characterization of photoreceptors and light-dependent processes described in this review, such as plastid signalling and diel regulation, is unveiling sensing systems which are unique to these algae, reflecting their complex evolutionary history and adaptation to aquatic life. Here, we also describe putative sensing components involved in the responses to nutrient, osmotic changes, and fluid motions. Continued elucidation of the molecular systems processing endogenous and environmental cues and their interactions with other biotic and abiotic stress signalling pathways is expected to greatly increase our understanding of the mechanisms controlling the abundance and distribution of the highly diverse diatom communities in marine ecosystems.
The aureochrome photoreceptor PtAUREO1a is a highly effective blue light switch in diatoms
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.