Impaired photoprotection in Phaeodactylum tricornutum KEA3 mutants reveals the proton regulatory circuit of diatoms light acclimation.

Diatoms are successful phytoplankton clades able to acclimate to changing environmental conditions, including e.g. variable light intensity. Diatoms are outstanding at dissipating light energy exceeding the maximum photosynthetic electron transfer (PET) capacity via the nonphotochemical quenching (NPQ) process. While the molecular effectors of NPQ as well as the involvement of the proton motive force (PMF) in its regulation are known, the regulators of the PET/PMF relationship remain unidentified in diatoms. We generated mutants of the H+ /K+ antiporter KEA3 in the model diatom Phaeodactylum tricornutum. Loss of KEA3 activity affects the PET/PMF coupling and NPQ responses at the onset of illumination, during transients and in steady-state conditions. Thus, this antiporter is a main regulator of the PET/PMF coupling. Consistent with this conclusion, a parsimonious model including only two free components, KEA3 and the diadinoxanthin de-epoxidase, describes most of the feedback loops between PET and NPQ. This simple regulatory system allows for efficient responses to fast (minutes) or slow (e.g. diel) changes in light environment, thanks to the presence of a regulatory calcium ion (Ca2+ )-binding domain in KEA3 modulating its activity. This circuit is likely tuned by the NPQ-effector proteins, LHCXs, providing diatoms with the required flexibility to thrive in different ocean provinces.

The fine-tuning of NPQ in diatoms relies on the regulation of both xanthophyll cycle enzymes.

Diatoms possess an efficient mechanism to dissipate photons as heat in conditions of excess light, which is visualized as the Non-Photochemical Quenching of chlorophyll a fluorescence (NPQ). In most diatom species, NPQ is proportional to the concentration of the xanthophyll cycle pigment diatoxanthin formed from diadinoxanthin by the diadinoxanthin de-epoxidase enzyme. The reverse reaction is performed by the diatoxanthin epoxidase. Despite the xanthophyll cycle's central role in photoprotection, its regulation is not yet well understood. The proportionality between diatoxanthin and NPQ allowed us to calculate the activity of both xanthophyll cycle enzymes in the model diatom Phaeodactylum tricornutum from NPQ kinetics. From there, we explored the light-dependency of the activity of both enzymes. Our results demonstrate that a tight regulation of both enzymes is key to fine-tune NPQ: (i) the rate constant of diadinoxanthin de-epoxidation is low under a light-limiting regime but increases as photosynthesis saturates, probably due to the thylakoidal proton gradient ΔpH (ii) the rate constant of diatoxanthin epoxidation exhibits an optimum under low light and decreases in the dark due to an insufficiency of the co-factor NADPH as well as in higher light through an as yet unresolved inhibition mechanism, that is unlikely to be related to the ΔpH. We observed that the suppression of NPQ by an uncoupler was due to an accelerated diatoxanthin epoxidation enzyme rather than to the usually hypothesized inhibition of the diadinoxanthin de-epoxidation enzyme.

Host specificity in diatom-bacteria interactions alleviates antagonistic effects.

While different microalgae tend to be associated with different bacteria, it remains unclear whether such specific associations are beneficial for the microalgae. We assessed the impact of bacterial isolates, derived from various marine benthic diatoms, on the growth of several strains belonging to the Cylindrotheca closterium diatom species complex. We first tested the effect of 35 different bacterial isolates on the growth of a single C. closterium strain, and then evaluated the impact of 8 of these isolates on the growth of 6 C. closterium strains and 1 Cylindrotheca fusiformis strain. Surprisingly, most interactions were neutral to antagonistic. The interactions were highly specific, with diatom growth in the presence of specific bacteria differing between Cylindrotheca strains and species, and closely related bacteria eliciting contrasting diatom growth responses. These differences could be related to the origin of the bacterial isolates, as only isolates from foreign diatom hosts significantly reduced diatom growth, implying coadaptation between different Cylindrotheca strains and their associated bacteria. Interestingly, the antagonistic effect of a Marinobacter strain was alleviated by the presence of a microbial inoculum that was native to the diatom host, suggesting that coadapted bacteria might also benefit their host indirectly by preventing the establishment of harmful bacteria.

N-Acyl Homoserine Lactone Derived Tetramic Acids Impair Photosynthesis in Phaeodactylum tricornutum.

Marine bacteria contribute substantially to nutrient cycling in the oceans and can engage in close interactions with microalgae. Many microalgae harbor characteristic satellite bacteria, many of which participate in N-acyl homoserine lactone (AHL) mediated quorum sensing. In the diffusion-controlled phycosphere, AHLs can reach high local concentrations, with some of them transforming into tetramic acids, compounds with a broad bioactivity. We tested a representative AHL, N-(3-oxododecanoyl) homoserine lactone, and its tetramic acid rearrangement product on the diatom Phaeodactylum tricornutum. While cell growth and photosynthetic efficiency of photosystem II were barely affected by the AHL, exposure to its tetramic acid rearrangement product had a negative effect on photosynthetic efficiency and led to growth inhibition and cell death in the long term, with a minimum inhibitory concentration between 20 and 50 µΜ. These results strengthen the view that AHLs may play an important role in shaping the outcome of microalgae-bacteria interactions.

Auxin Function in the Brown Alga Dictyota dichotoma.

Auxin controls body plan patterning in land plants and has been proposed to play a similar role in the development of brown algae (Phaeophyta) despite their distant evolutionary relationship with land plants. The mechanism of auxin action in brown algae remains controversial because of contradicting conclusions derived from pharmacological studies on Fucus In this study, we used Dictyota dichotoma as a model system to show that auxin plays a role during the apical-basal patterning of the embryo of brown algae. Indole-3-acetic acid was detectable in D. dichotoma germlings and mature tissue. Although two-celled D. dichotoma zygotes normally develop a rhizoid from one pole and a thallus meristem from the other, addition of exogenous auxins to one-celled embryos affected polarization, and both poles of the spheroidal embryo developed into rhizoids instead. The effect was strongest at lower pH and when variable extrinsic informational cues were applied. 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid, an inhibitor of the ABC-B/multidrug resistance/P-glycoprotein subfamily of transporters in land plants, affected rhizoid formation by increasing rhizoid branching and inducing ectopic rhizoids. An in silico survey of auxin genes suggested that a diverse range of biosynthesis genes and transport genes, such as PIN-LIKES, and the ATP-binding cassette subfamily (ABC-B/multidrug resistance/P-glycoprotein) transporters from land plants have homologs in D. dichotoma and Ectocarpus siliculosus Together with reports on auxin function in basal lineages of green algae, these results suggest that auxin function predates the divergence between the green and brown lineage and the transition toward land plants.

Nematodes stimulate biomass accumulation in a multispecies diatom biofilm.

While the effects of abiotic parameters on microbial tidal biofilms are relatively well-documented, the effects of grazing and/or bioturbation by meiofauna are poorly understood. We investigated the impact of a natural nematode assemblage on the biomass and microbial community structure of a multispecies diatom biofilm. Nematodes stimulated diatom biomass accumulation of the biofilm and caused a shift in diatom community structure. Higher diatom biomass accumulation in the presence of nematodes could be the result of increased diatom biomass production through nutrient regeneration resulting from grazing or bioturbation, and/or through shifts in interspecific interactions between diatoms (e.g. competition) through selective grazing. Alternatively, lower biomass in the controls may be due to higher secretion of diatom production in the form of bound extracellular polymeric substances (EPS). Our observations underscore that meiobenthos, and especially nematodes, are important for the structure and production of tidal biofilms.

Expanding the toolbox for cryopreservation of marine and freshwater diatoms.

Diatoms constitute the most diverse group of microalgae and have long been recognised for their large biotechnological potential. In the wake of growing research interest in new model species and development of commercial applications, there is a pressing need for long-term preservation of diatom strains. While cryopreservation using dimethylsulfoxide (DMSO) as a cryoprotective agent is the preferred method for long-term strain preservation, many diatom species cannot be successfully cryopreserved using DMSO. Therefore, in this study, we studied cryopreservation success in six different diatom species, representing the major morphological and ecological diatom groups, using a range of DMSO concentrations and Plant Vitrification Solution 2 (PVS2) as an alternative cryoprotectant to DMSO. In addition, we tested whether suppressing bacterial growth by antibiotics accelerates the post-thaw recovery process. Our results show that the effects of cryoprotectant choice, its concentration and the addition of antibiotics are highly species specific. In addition, we showed that PVS2 and antibiotics are useful agents to optimize cryopreservation of algae that cannot survive the traditional cryopreservation protocol using DMSO. We conclude that a species-specific approach will remain necessary to develop protocols for diatom cryopreservation and to increase their representation in public culture collections.

Growth form defines physiological photoprotective capacity in intertidal benthic diatoms.

In intertidal marine sediments, characterized by rapidly fluctuating and often extreme light conditions, primary production is frequently dominated by diatoms. We performed a comparative analysis of photophysiological traits in 15 marine benthic diatom species belonging to the four major morphological growth forms (epipelon (EPL), motile epipsammon (EPM-M) and non-motile epipsammon (EPM-NM) and tychoplankton (TYCHO)) found in these sediments. Our analyses revealed a clear relationship between growth form and photoprotective capacity, and identified fast regulatory physiological photoprotective traits (that is, non-photochemical quenching (NPQ) and the xanthophyll cycle (XC)) as key traits defining the functional light response of these diatoms. EPM-NM and motile EPL showed the highest and lowest NPQ, respectively, with EPM-M showing intermediate values. Like EPL, TYCHO had low NPQ, irrespective of whether they were grown in benthic or planktonic conditions, reflecting an adaptation to a low light environment. Our results thus provide the first experimental evidence for the existence of a trade-off between behavioural (motility) and physiological photoprotective mechanisms (NPQ and the XC) in the four major intertidal benthic diatoms growth forms using unialgal cultures. Remarkably, although motility is restricted to the raphid pennate diatom clade, raphid pennate species, which have adopted a non-motile epipsammic or a tychoplanktonic life style, display the physiological photoprotective response typical of these growth forms. This observation underscores the importance of growth form and not phylogenetic relatedness as the prime determinant shaping the physiological photoprotective capacity of benthic diatoms.

Haloperoxidase mediated quorum quenching by Nitzschia cf pellucida: study of the metabolization of N-acyl homoserine lactones by a benthic diatom.

Diatoms are known to produce a variety of halogenated compounds, which were recently shown to have a role in allelopathic interactions between competing species. The production of these compounds is linked to haloperoxidase activity. This research, has shown that this system may also be involved in diatom-bacteria interactions via the H2O2 dependent inactivation of a type of quorum sensing (QS) molecule, i.e., N-ß-ketoacylated homoserine lactones (AHLs), by a natural haloperoxidase system from the benthic diatom Nitzschia cf pellucida. The AHL degradation pathway towards corresponding halogenated derivatives was elucidated via HPLC-MS analysis and the synthesis of a broad series of novel halogenated AHL analogues as reference compounds. Furthermore, their biological activity as quorum sensing modulators was directly compared and evaluated against a series of naturally occurring ß-keto-AHLs. It has been demonstrated that the loss of the QS activity results from the final cleavage of the halogenated N-acyl chain of the signal molecules.