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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

PII Overexpression in Lotus japonicus Affects Nodule Activity in Permissive Low-Nitrogen Conditions and Increases Nodule Numbers in High Nitrogen Treated Plants.

We report here the first characterization of a GLNB1 gene coding for the PII protein in leguminous plants. The main purpose of this work was the investigation of the possible roles played by this multifunctional protein in nodulation pathways. The Lotus japonicus LjGLB1 gene shows a significant transcriptional regulation during the light-dark cycle and different nitrogen availability, conditions that strongly affect nodule formation, development, and functioning. We also report analysis of the spatial profile of expression of LjGLB1 in root and nodule tissues and of the protein's subcellular localization. Transgenic L. japonicus lines overexpressing the PII protein were obtained and tested for the analysis of the symbiotic responses in different conditions. The uncoupling of PII from its native regulation affects nitrogenase activity and nodule polyamine content. Furthermore, our results suggest the involvement of PII in the signaling of the nitrogen nutritional status affecting the legumes' predisposition for nodule formation.

The diversity of small non-coding RNAs in the diatom Phaeodactylum tricornutum.

BACKGROUND: Marine diatoms constitute a major component of eukaryotic phytoplankton and stand at the crossroads of several evolutionary lineages. These microalgae possess peculiar genomic features and novel combinations of genes acquired from bacterial, animal and plant ancestors. Furthermore, they display both DNA methylation and gene silencing activities. Yet, the biogenesis and regulatory function of small RNAs (sRNAs) remain ill defined in diatoms. RESULTS: Here we report the first comprehensive characterization of the sRNA landscape and its correlation with genomic and epigenomic information in Phaeodactylum tricornutum. The majority of sRNAs is 25 to 30 nt-long and maps to repetitive and silenced Transposable Elements marked by DNA methylation. A subset of this population also targets DNA methylated protein-coding genes, suggesting that gene body methylation might be sRNA-driven in diatoms. Remarkably, 25-30 nt sRNAs display a well-defined and unprecedented 180 nt-long periodic distribution at several highly methylated regions that awaits characterization. While canonical miRNAs are not detectable, other 21-25 nt sRNAs of unknown origin are highly expressed. Besides, non-coding RNAs with well-described function, namely tRNAs and U2 snRNA, constitute a major source of 21-25 nt sRNAs and likely play important roles under stressful environmental conditions. CONCLUSIONS: P. tricornutum has evolved diversified sRNA pathways, likely implicated in the regulation of largely still uncharacterized genetic and epigenetic processes. These results uncover an unexpected complexity of diatom sRNA population and previously unappreciated features, providing new insights into the diversification of sRNA-based processes in eukaryotes.

High light acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool.

In diatoms, the process of energy-dependent chlorophyll fluorescence quenching (qE) has an important role in photoprotection. Three components are essential for qE: (1) the light-dependent generation of a transthylakoidal proton gradient; (2) the deepoxidation of the xanthophyll diadinoxanthin (Dd) into diatoxanthin (Dt); and (3) specific nucleus-encoded antenna proteins, called Light Harvesting Complex Protein X (LHCX). We used the model diatom Phaeodactylum tricornutum to investigate the concerted light acclimation response of the qE key components LHCX, proton gradient, and xanthophyll cycle pigments (Dd+Dt) and to identify the intracellular light-responsive trigger. At high-light exposure, the up-regulation of three of the LHCX genes and the de novo synthesis of Dd+Dt led to a pronounced rise of qE. By inhibiting either the conversion of Dd to Dt or the translation of LHCX genes, qE amplification was abolished and the diatom cells suffered from stronger photoinhibition. Artificial modification of the redox state of the plastoquinone (PQ) pool via 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone resulted in a disturbance of Dd+Dt synthesis in an opposite way. Moreover, we could increase the transcription of two of the four LHCX genes under low-light conditions by reducing the PQ pool using 5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone. Altogether, our results underline the central role of the redox state of the PQ pool in the light acclimation of diatoms. Additionally, they emphasize strong evidence for the existence of a plastid-to-nucleus retrograde signaling mechanism in an organism with plastids that derived from secondary endosymbiosis.

An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light.

Diatoms are prominent phytoplanktonic organisms that contribute around 40% of carbon assimilation in the oceans. They grow and perform optimally in variable environments, being able to cope with unpredictable changes in the amount and quality of light. The molecular mechanisms regulating diatom light responses are, however, still obscure. Using knockdown Phaeodactylum tricornutum transgenic lines, we reveal the key function of a member of the light-harvesting complex stress-related (LHCSR) protein family, denoted LHCX1, in modulation of excess light energy dissipation. In contrast to green algae, this gene is already maximally expressed in nonstressful light conditions and encodes a protein required for efficient light responses and growth. LHCX1 also influences natural variability in photoresponse, as evidenced in ecotypes isolated from different latitudes that display different LHCX1 protein levels. We conclude, therefore, that this gene plays a pivotal role in managing light responses in diatoms.

Exploring the molecular basis of responses to light in marine diatoms.

Light is an essential source of energy for life on Earth and is one of the most important signals that organisms use to obtain information from the surrounding environment, on land and in the oceans. Prominent marine microalgae, such as diatoms, display a suite of sophisticated responses (physiological, biochemical, and behavioural) to optimize their photosynthesis and growth under changing light conditions. However, the molecular mechanisms controlling diatom responses to light are still largely unknown. Recent progress in marine diatom genomics and genetics, combined with well-established (eco) physiological and biophysical approaches, now offers novel opportunities to address these issues. This review provides a description of the molecular components identified in diatom genomes that are involved in light perception and acclimation mechanisms. How the initial functional characterizations of specific light regulators provide the basis to investigate the conservation or diversification of light-mediated processes in diatoms is also discussed. Hypotheses on the role of the identified factors in determining the growth, distribution, and adaptation of diatoms in different marine environments are reported.

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.

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.

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.