The tonoplast localized protein PtNPF1 participates in the regulation of nitrogen response in diatoms.

Diatoms are a highly successful group of phytoplankton, well adapted also to oligotrophic environments and capable of handling nutrient fluctuations in the ocean, particularly nitrate. The presence of a large vacuole is an important trait contributing to their adaptive features. It confers diatoms the ability to accumulate and store nutrients, such as nitrate, when they are abundant outside and then to reallocate them into the cytosol to meet deficiencies, in a process called luxury uptake. The molecular mechanisms that regulate these nitrate fluxes are still not known in diatoms. In this work, we provide new insights into the function of Phaeodactylum tricornutum NPF1, a putative low-affinity nitrate transporter. To accomplish this, we generated overexpressing strains and CRISPR/Cas9 loss-of-function mutants. Microscopy observations confirmed predictions that PtNPF1 is localized on the vacuole membrane. Furthermore, functional characterizations performed on knock-out mutants revealed a transient growth delay phenotype linked to altered nitrate uptake. Together, these results allowed us to hypothesize that PtNPF1 is presumably involved in modulating intracellular nitrogen fluxes, managing intracellular nutrient availability. This ability might allow diatoms to fine-tune the assimilation, storage and reallocation of nitrate, conferring them a strong advantage in oligotrophic environments.

Phaeodactylum tricornutum: An established model species for diatom molecular research and an emerging chassis for algal synthetic biology.

Diatoms are prominent and highly diverse microalgae in aquatic environments. Compared with other diatom species, Phaeodactylum tricornutum is an "atypical diatom" displaying three different morphotypes and lacking the usual silica shell. Despite being of limited ecological relevance, its ease of growth in the laboratory and well-known physiology, alongside the steady increase in genome-enabled information coupled with effective tools for manipulating gene expression, have meant it has gained increased recognition as a powerful experimental model for molecular research on diatoms. We here present a brief overview of how over the last 25 years P. tricornutum has contributed to the unveiling of fundamental aspects of diatom biology, while also emerging as a new tool for algal process engineering and synthetic biology.

Gene expression during the formation of resting spores induced by nitrogen starvation in the marine diatom Chaetoceros socialis.

BACKGROUND: Dormancy is widespread in both multicellular and unicellular organisms. Among diatoms, unicellular microalgae at the base of all aquatic food webs, several species produce dormant cells (spores or resting cells) that can withstand long periods of adverse environmental conditions. RESULTS: We present the first gene expression study during the process of spore formation induced by nitrogen depletion in the marine planktonic diatom Chaetoceros socialis. In this condition, genes related to photosynthesis and nitrate assimilation, including high-affinity nitrate transporters (NTRs), were downregulated. While the former result is a common reaction among diatoms under nitrogen stress, the latter seems to be exclusive of the spore-former C. socialis. The upregulation of catabolic pathways, such as tricarboxylic acid cycle, glyoxylate cycle and fatty acid beta-oxidation, suggests that this diatom could use lipids as a source of energy during the process of spore formation. Furthermore, the upregulation of a lipoxygenase and several aldehyde dehydrogenases (ALDHs) advocates the presence of oxylipin-mediated signaling, while the upregulation of genes involved in dormancy-related pathways conserved in other organisms (e.g. serine/threonine-protein kinases TOR and its inhibitor GATOR) provides interesting avenues for future explorations. CONCLUSIONS: Our results demonstrate that the transition from an active growth phase to a resting one is characterized by marked metabolic changes and provides evidence for the presence of signaling pathways related to intercellular communication.

LjNRT2.3 plays a hierarchical role in the control of high affinity transport system for root nitrate acquisition in Lotus japonicus.

Nitrate is a key mineral nutrient required for plant growth and development. Plants have evolved sophisticated mechanisms to respond to changes of nutritional availability in the surrounding environment and the optimization of root nitrate acquisition under nitrogen starvation is crucial to cope with unfavoured condition of growth. In this study we present a general description of the regulatory transcriptional and spatial profile of expression of the Lotus japonicus nitrate transporter NRT2 family. Furthermore, we report a phenotypic characterization of two independent Ljnrt2.3 knock out mutants indicating the involvement of the LjNRT2.3 gene in the root nitrate acquisition and lateral root elongation pathways occurring in response to N starvation conditions. We also report an epistatic relationship between LjNRT2.3 and LjNRT2.1 suggesting a combined mode of action of these two genes in order to optimize the Lotus response to a prolonged N starvation.

Detection and Quantification of Small Noncoding RNAs in Marine Diatoms.

Endogenous small noncoding RNAs (sRNAs) are a large family of essential regulators of gene expression in both eukaryotes and prokaryotes. Various types of sRNAs with different size and mapping to different genome locations have been recently identified in diatoms, a successful group of phytoplankton in the marine environment. However, their biogenesis and regulatory function are still largely unknown and unexplored in these microalgae, also due to the lack of methods for their experimental analysis. Herein, we present a point-by-point description of the protocols for detection and quantification of sRNAs by Northern-blot analysis and quantitative stem-loop RT-PCR, established in the diatom molecular model specie Phaeodactylum tricornutum.

Optimized Proteolistic Protocol for the Delivery of the Cas9 Protein in Phaeodactylum tricornutum.

The CRISPR/Cas9 system coupled with proteolistics is a DNA-free nuclear transformation method based on the introduction of ribonucleoprotein (RNP) complexes into cells. The method has been set up for diatoms as an alternative to genetic transformation via biolistics and has the advantages of reducing off-target mutations, limiting the working time of the Cas9 endonuclease, and overcoming the occurrence of random insertions of the transgene in the genome. We present a point-by-point description of the protocol with modifications that make it more cost-effective, by reducing the amount of the enzyme while maintaining a comparable efficiency to the original protocol, and with an increased concentration of the selective drug which allows to reduce false positives.

The Lotus japonicus AFB6 Gene Is Involved in the Auxin Dependent Root Developmental Program.

Auxin is essential for root development, and its regulatory action is exerted at different steps from perception of the hormone up to transcriptional regulation of target genes. In legume plants there is an overlap between the developmental programs governing lateral root and N2-fixing nodule organogenesis, the latter induced as the result of the symbiotic interaction with rhizobia. Here we report the characterization of a member of the L. japonicus TIR1/AFB auxin receptor family, LjAFB6. A preferential expression of the LjAFB6 gene in the aerial portion of L. japonicus plants was observed. Significant regulation of the expression was not observed during the symbiotic interaction with Mesorhizobium loti and the nodule organogenesis process. In roots, the LjAFB6 expression was induced in response to nitrate supply and was mainly localized in the meristematic regions of both primary and lateral roots. The phenotypic analyses conducted on two independent null mutants indicated a specialized role in the control of primary and lateral root elongation processes in response to auxin, whereas no involvement in the nodulation process was found. We also report the involvement of LjAFB6 in the hypocotyl elongation process and in the control of the expression profile of an auxin-responsive gene.

The Lotus japonicus NPF3.1 Is a Nodule-Induced Gene That Plays a Positive Role in Nodule Functioning.

Nitrogen-fixing nodules are new organs formed on legume roots as a result of the beneficial interaction with the soil bacteria, rhizobia. Proteins of the nitrate transporter 1/peptide transporter family (NPF) are largely represented in the subcategory of nodule-induced transporters identified in mature nodules. The role of nitrate as a signal/nutrient regulating nodule functioning has been recently highlighted in the literature, and NPFs may play a central role in both the permissive and inhibitory pathways controlling N2-fixation efficiency. In this study, we present the characterization of the Lotus japonicus LjNPF3.1 gene. LjNPF3.1 is upregulated in mature nodules. Promoter studies show transcriptional activation confined to the cortical region of both roots and nodules. Under symbiotic conditions, Ljnpf3.1-knockout mutant's display reduced shoot development and anthocyanin accumulation as a result of nutrient deprivation. Altogether, LjNPF3.1 plays a role in maximizing the beneficial outcome of the root nodule symbiosis.

Integrative omics identification, evolutionary and structural analysis of low affinity nitrate transporters in diatoms, diNPFs.

Diatoms are one of the major and most diverse groups of phytoplankton, with chimeric genomes harbouring a combination of genes of bacterial, animal and plant origin. They have developed sophisticated mechanisms to face environmental variations. In marine environments, nutrients concentration shows significant temporal and spatial variability, influencing phytoplankton growth. Among nutrients, nitrogen, present at micromolar levels, is often a limiting resource. Here, we report a comprehensive characterization of the Nitrate Transporter 1/Peptide Transporter Family (NPF) in diatoms, diNPFs. NPFs are well characterized in many organisms where they recognize a broad range of substrates, ranging from short-chained di- and tri-peptides in bacteria, fungi and mammals to a wide variety of molecules including nitrate in higher plants. Scarce information is available for diNPFs. We integrated-omics, phylogenetic, structural and expression analyses, to infer information on their role in diatoms. diNPF genes diverged to produce two distinct clades with strong sequence and structural homology with either bacterial or plant NPFs, with different predicted sub-cellular localization, suggesting that the divergence resulted in functional diversification. Moreover, transcription analysis of diNPF genes under different laboratory and environmental growth conditions suggests that diNPF diversification led to genetic adaptations that might contribute to diatoms ability to flourish in diverse environmental conditions.

The functional characterization of LjNRT2.4 indicates a novel, positive role of nitrate for an efficient nodule N2 -fixation activity.

Atmospheric nitrogen (N2) -fixing nodules are formed on the roots of legume plants as result of the symbiotic interaction with rhizobia. Nodule functioning requires high amounts of carbon and energy, and therefore legumes have developed finely tuned mechanisms to cope with changing external environmental conditions, including nutrient availability and flooding. The investigation of the role of nitrate as regulator of the symbiotic N2 fixation has been limited to the inhibitory effects exerted by high external concentrations on nodule formation, development and functioning. We describe a nitrate-dependent route acting at low external concentrations that become crucial in hydroponic conditions to ensure an efficient nodule functionality. Combined genetic, biochemical and molecular studies are used to unravel the novel function of the LjNRT2.4 gene. Two independent null mutants are affected by the nitrate content of nodules, consistent with LjNRT2.4 temporal and spatial profiles of expression. The reduced nodular nitrate content is associated to a strong reduction of nitrogenase activity and a severe N-starvation phenotype observed under hydroponic conditions. We also report the effects of the mutations on the nodular nitric oxide (NO) production and content. We discuss the involvement of LjNRT2.4 in a nitrate-NO respiratory chain taking place in the N2 -fixing nodules.

Disruption of the Lotus japonicus transporter LjNPF2.9 increases shoot biomass and nitrate content without affecting symbiotic performances.

BACKGROUND: After uptake from soil into the root tissue, distribution and allocation of nitrate throughout the whole plant body, is a critical step of nitrogen use efficiency (NUE) and for modulation of plant growth in response to various environmental conditions. In legume plants nitrate distribution is also important for the regulation of the nodulation process that allows to fix atmospheric N (N2) through the symbiotic interaction with rhizobia (symbiotic nitrogen fixation, SNF). RESULTS: Here we report the functional characterization of the Lotus japonicus gene LjNPF2.9, which is expressed mainly in the root vascular structures, a key localization for the control of nitrate allocation throughout the plant body. LjNPF2.9 expression in Xenopus laevis oocytes induces 15NO3 accumulation indicating that it functions as a nitrate importer. The phenotypic characterization of three independent knock out mutants indicates an increased shoot biomass in the mutant backgrounds. This phenotype is associated to an increased/decreased nitrate content detected in the shoots/roots. Furthermore, our analysis indicates that the accumulation of nitrate in the shoot does not affect the nodulation and N-Fixation capacities of the knock out mutants. CONCLUSIONS: This study shows that LjNPF2.9 plays a crucial role in the downward transport of nitrate to roots, occurring likely through a xylem-to-phloem loading-mediated activity. The increase of the shoot biomass and nitrate accumulation might represent a relevant phenotype in the perspective of an improved NUE and this is further reinforced in legume plants by the reported lack of effects on the SNF efficiency.

Meta-Omics Reveals Genetic Flexibility of Diatom Nitrogen Transporters in Response to Environmental Changes.

Diatoms (Bacillariophyta), one of the most abundant and diverse groups of marine phytoplankton, respond rapidly to the supply of new nutrients, often out-competing other phytoplankton. Herein, we integrated analyses of the evolution, distribution, and expression modulation of two gene families involved in diatom nitrogen uptake (DiAMT1 and DiNRT2), in order to infer the main drivers of divergence in a key functional trait of phytoplankton. Our results suggest that major steps in the evolution of the two gene families reflected key events triggering diatom radiation and diversification. Their expression is modulated in the contemporary ocean by seawater temperature, nitrate, and iron concentrations. Moreover, the differences in diversity and expression of these gene families throughout the water column hint at a possible link with bacterial activity. This study represents a proof-of-concept of how a holistic approach may shed light on the functional biology of organisms in their natural environment.

Marine Natural Products from Microalgae: An -Omics Overview.

Over the last decade, genome sequences and other -omics datasets have been produced for a wide range of microalgae, and several others are on the way. Marine microalgae possess distinct and unique metabolic pathways, and can potentially produce specific secondary metabolites with biological activity (e.g., antipredator, allelopathic, antiproliferative, cytotoxic, anticancer, photoprotective, as well as anti-infective and antifouling activities). Because microalgae are very diverse, and adapted to a broad variety of environmental conditions, the chances to find novel and unexplored bioactive metabolites with properties of interest for biotechnological and biomedical applications are high. This review presents a comprehensive overview of the current efforts and of the available solutions to produce, explore and exploit -omics datasets, with the aim of identifying species and strains with the highest potential for the identification of novel marine natural products. In addition, funding efforts for the implementation of marine microalgal -omics resources and future perspectives are presented as well.

Phaeodactylum tricornutum as a model organism for testing the membrane penetrability of sulphonamide carbonic anhydrase inhibitors.

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes, which started to be investigated in detail in pathogenic, as well as non-pathogenic species since their pivotal role is to accelerate the physiological CO2 hydration/dehydration reaction significantly. Here, we propose the marine unicellular diatom Phaeodactylum tricornutum as a model organism for testing the membrane penetrability of CA inhibitors (CAIs). Seven inhibitors belonging to the sulphonamide type and possessing a diverse scaffold have been explored for their in vitro inhibition of the whole diatom CAs and the in vivo inhibitory effect on the growth of P. tricornutum. Interesting, inhibition of growth was observed, in vivo, demonstrating that this diatom is a good model for testing the cell wall penetrability of this class of pharmacological agents. Considering that many pathogens are difficult and dangerous to grow in the laboratory, the growth inhibition of P. tricornutum with different such CAIs may be subsequently used to design inhibition studies of CAs from pathogenic organisms.

Bioderived Three-Dimensional Hierarchical Nanostructures as Efficient Surface-Enhanced Raman Scattering Substrates for Cell Membrane Probing.

In this work, we propose the use of complex, bioderived nanostructures as efficient surface-enhanced Raman scattering (SERS) substrates for chemical analysis of cellular membranes. These structures were directly obtained from a suitable gold metalization of the Pseudonitzchia multistriata diatom silica shell (the so called frustule), whose grating-like geometry provides large light coupling with external radiation, whereas its extruded, subwavelength lateral edge provides an excellent interaction with cells without steric hindrance. We carried out numerical simulations and experimental characterizations of the supported plasmonic resonances and optical near-field amplification. We thoroughly evaluated the SERS substrate enhancement factor as a function of the metalization parameters and finally applied the nanostrucures for discriminating cell membrane Raman signals. In particular, we considered two cases where the membrane composition plays a fundamental role in the assessment of several pathologies, that is, red blood cells and B-leukemia REH cells.

The Nitrate Transporter Family Protein LjNPF8.6 Controls the N-Fixing Nodule Activity.

N-fixing nodules are new organs formed on legume roots as a result of the beneficial interaction with soil bacteria, rhizobia. The nodule functioning is still a poorly characterized step of the symbiotic interaction, as only a few of the genes induced in N-fixing nodules have been functionally characterized. We present here the characterization of a member of the Lotus japonicus nitrate transporter1/peptide transporter family, LjNPF8.6 The phenotypic characterization carried out in independent L. japonicus LORE1 insertion lines indicates a positive role of LjNPF8.6 on nodule functioning, as knockout mutants display N-fixation deficiency (25%) and increased nodular superoxide content. The partially compromised nodule functioning induces two striking phenotypes: anthocyanin accumulation already displayed 4 weeks after inoculation and shoot biomass deficiency, which is detected by long-term phenotyping. LjNPF8.6 achieves nitrate uptake in Xenopus laevis oocytes at both 0.5 and 30 mm external concentrations, and a possible role as a nitrate transporter in the control of N-fixing nodule activity is discussed.

Diatom Frustule Morphogenesis and Function: a Multidisciplinary Survey.

Diatoms represent the major component of phytoplankton and are responsible for about 20-25% of global primary production. Hundreds of millions of years of evolution led to tens of thousands of species differing in dimensions and morphologies. In particular, diatom porous silica cell walls, the frustules, are characterized by an extraordinary, species-specific diversity. It is of great interest, among the marine biologists and geneticists community, to shed light on the origin and evolutionary advantage of this variability of dimensions, geometries and pore distributions. In the present article the main reported data related to frustule morphogenesis and functionalities with contributions from fundamental biology, genetics, mathematics, geometry and physics are reviewed.

The diatom Phaeodactylum tricornutum adjusts nonphotochemical fluorescence quenching capacity in response to dynamic light via fine-tuned Lhcx and xanthophyll cycle pigment synthesis.

Diatoms contain a highly flexible capacity to dissipate excessively absorbed light by nonphotochemical fluorescence quenching (NPQ) based on the light-induced conversion of diadinoxanthin (Dd) into diatoxanthin (Dt) and the presence of Lhcx proteins. Their NPQ fine regulation on the molecular level upon a shift to dynamic light conditions is unknown. We investigated the regulation of Dd + Dt amount, Lhcx gene and protein synthesis and NPQ capacity in the diatom Phaeodactylum tricornutum after a change from continuous low light to 3 d of sine (SL) or fluctuating (FL) light conditions. Four P. tricornutum strains with different NPQ capacities due to different expression of Lhcx1 were included. All strains responded to dynamic light comparably, independently of initial NPQ capacity. During SL, NPQ capacity was strongly enhanced due to a gradual increase of Lhcx2 and Dd + Dt amount. During FL, cells enhanced their NPQ capacity on the first day due to increased Dd + Dt, Lhcx2 and Lhcx3; already by the second day light acclimation was accomplished. While quenching efficiency of Dt was strongly lowered during SL conditions, it remained high throughout the whole FL exposure. Our results highlight a more balanced and cost-effective photoacclimation strategy of P. tricornutum under FL than under SL conditions.

Multisignal control of expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum.

Diatoms are phytoplanktonic organisms that grow successfully in the ocean where light conditions are highly variable. Studies of the molecular mechanisms of light acclimation in the marine diatom Phaeodactylum tricornutum show that carotenoid de-epoxidation enzymes and LHCX1, a member of the light-harvesting protein family, both contribute to dissipate excess light energy through non-photochemical quenching (NPQ). In this study, we investigate the role of the other members of the LHCX family in diatom stress responses. Our analysis of available genomic data shows that the presence of multiple LHCX genes is a conserved feature of diatom species living in different ecological niches. Moreover, an analysis of the levels of four P. tricornutum LHCX transcripts in relation to protein expression and photosynthetic activity indicates that LHCXs are differentially regulated under different light intensities and nutrient starvation, mostly modulating NPQ capacity. We conclude that multiple abiotic stress signals converge to regulate the LHCX content of cells, providing a way to fine-tune light harvesting and photoprotection. Moreover, our data indicate that the expansion of the LHCX gene family reflects functional diversification of its members which could benefit cells responding to highly variable ocean environments.

Down-regulated Lotus japonicus GCR1 plants exhibit nodulation signalling pathways alteration.

G Protein Coupled Receptor (GPCRs) are integral membrane proteins involved in various signalling pathways by perceiving many extracellular signals and transducing them to heterotrimeric G proteins, which further transduce these signals to intracellular downstream effectors. GCR1 is the only reliable plant candidate as a member of the GPCRs superfamily. In the legume/rhizobia symbiotic interaction, G proteins are involved in signalling pathways controlling different steps of the nodulation program. In order to investigate the putative hierarchic role played by GCR1 in these symbiotic pathways we identified and characterized the Lotus japonicus gene encoding the seven transmembrane GCR1 protein. The detailed molecular and topological analyses of LjGCR1 expression patterns that are presented suggest a possible involvement in the early steps of nodule organogenesis. Furthermore, phenotypic analyses of independent transgenic RNAi lines, showing a significant LjGCR1 expression down regulation, suggest an epistatic action in the control of molecular markers of nodulation pathways, although no macroscopic symbiotic phenotypes could be revealed.