Alessandra Rogato

Gene silencing in the marine diatom Phaeodactylum tricornutum

Diatoms are a major but poorly understood phytoplankton group. The recent completion of two whole genome sequences has revealed that they contain unique combinations of genes, likely recruited during their history as secondary endosymbionts, as well as by horizontal gene transfer from bacteria. A major limitation for the study of diatom biology and gene function is the lack of tools to generate targeted gene knockout or knockdown mutants. In this work, we have assessed the possibility of triggering gene silencing in Phaeodactylum tricornutum using constructs containing either anti-sense or inverted repeat sequences of selected target genes. We report the successful silencing of a GUS reporter gene expressed in transgenic lines, as well as the knockdown of endogenous phytochrome (DPH1) and cryptochrome (CPF1) genes. To highlight the utility of the approach we also report the first phenotypic characterization of a diatom mutant (cpf1). Our data open the way for reverse genetics in diatoms and represent a major advance for understanding their biology and ecology. Initial molecular analyses reveal that targeted downregulation likely occurs through transcriptional and post-transcriptional gene silencing mechanisms. Interestingly, molecular players involved in RNA silencing in other eukaryotes are only poorly conserved in diatoms.

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.

Diatom PtCPF1 is a new cryptochrome/photolyase family member with DNA repair and transcription regulation activity

Members of the cryptochrome/photolyase family (CPF) are widely distributed throughout all kingdoms, and encode photosensitive proteins that typically show either photoreceptor or DNA repair activity. Animal and plant cryptochromes have lost DNA repair activity and now perform specialized photoperceptory functions, for example, plant cryptochromes regulate growth and circadian rhythms, whereas mammalian and insect cryptochromes act as transcriptional repressors that control the circadian clock. However, the functional differentiation between photolyases and cryptochromes is now being questioned. Here, we show that the PtCPF1 protein from the marine diatom Phaeodactylum tricornutum shows 6‐4 photoproduct repair activity and can act as a transcriptional repressor of the circadian clock in a heterologous mammalian cell system. Conversely, it seems to have a wide role in blue‐light‐regulated gene expression in diatoms. The protein might therefore represent a missing link in the evolution of CPFs, and act as a novel ultraviolet/blue light sensor in marine environments.

High Light Acclimation in the Secondary Plastids Containing Diatom Phaeodactylum tricornutum is Triggered by the Redox State of the Plastoquinone Pool

Summary: The redox state of the plastidic plastoquinone pool triggers a retrograde signal in diatoms, organisms with plastids evolved by secondary endosymbiosis. 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.

Characterization of Three Functional High-Affinity Ammonium Transporters in Lotus japonicus with Differential Transcriptional Regulation and Spatial Expression

Ammonium is a primary source of nitrogen for plants. In legume plants ammonium can also be obtained by symbiotic nitrogen fixation, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{{+}}\) \end{document} is also a regulator of early and late symbiotic interaction steps. Ammonium transporters are likely to play important roles in the control of nodule formation as well as in nitrogen assimilation. Two new genes, LjAMT1;2 and LjAMT1;3, were cloned from Lotus japonicus. Both were able to complement the growth defect of a yeast (Saccharomyces cerevisiae) ammonium transport mutant. Measurement of [14C]methylammonium uptake rates and competition experiments revealed that each transporter had a high affinity for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{{+}}\) \end{document}. The Ki for ammonium was 1.7, 3, and 15 μm for LjAMT1;1, 1;2, and 1;3, respectively. Real-time PCR revealed higher expression of LjAMT1;1, 1;2, and 1;3 genes in leaves than in roots and nodule, with expression levels decreasing in the order LjAMT1;1 > 1;2 > 1;3 except in flowers, in which LjAMT1;3 was expressed at higher level than in leaves, and LjAMT1;1 showed the lowest level of expression. Expression of LjAMT1;1 and 1;2 in roots was induced by nitrogen deprivation. Expression of LjAMT1;1 was repressed in leaves exposed to elevated CO2 concentrations, which also suppress photorespiration. Tissue and cellular localization of LjAMT1 genes expression, using promoter-β-glucuronidase and in situ RNA hybridization approaches, revealed distinct cellular spatial localization in different organs, including nodules, suggesting differential roles in the nitrogen metabolism of these organs.

Differential effects of combined N sources on early steps of the Nod factor-dependent transduction pathway in Lotus japonicus.

The development of nitrogen-fixing nodules in legumes is induced by perception of lipochitin-oligosaccharide signals secreted by a bacterial symbiont. Nitrogen (N) starvation is a prerequisite for the formation, development, and function of root nodules, and high levels of combined N in the form of nitrate or ammonium can completely abolish nodule formation. We distinguished between nitrate and ammonium inhibitory effects by identifying when and where these combined N sources interfere with the Nod-factor-induced pathway. Furthermore, we present a small-scale analysis of the expression profile, under different N conditions, of recently identified genes involved in the Nod-factor-induced pathway. In the presence of high levels of nitrate or ammonium, the NIN gene fails to be induced 24 h after the addition of Nod factor compared with plants grown under N-free conditions. This induction is restored in the hypernodulating nitrate-tolerant har1-3 mutant only in the presence of 10 and 20 mM KNO3. These results were confirmed in Lotus plants inoculated with Mesorhizobium loti. NIN plays a key role in the nodule organogenesis program and its downregulation may represent a crucial event in the nitrate-dependent pathway leading to the inhibition of nodule organogenesis.

Symbiotic competence in Lotus japonicus is affected by plant nitrogen status: transcriptomic identification of genes affected by a new signalling pathway

In leguminous plants, symbiotic nitrogen (N) fixation performances and N environmental conditions are linked because nodule initiation, development and functioning are greatly influenced by the amount of available N sources. We demonstrate here that N supply also controls, beforehand, the competence of leguminous plants to perform the nodulation program. Lotus japonicus plants preincubated for 10 d in high-N conditions, and then transferred to low N before the Mesorhizobium loti inoculation, had reduced nodulation. This phenotype was maintained for at least 6 d and a complete reacquisition of the symbiotic competence was observed only after 9 d. The time-course analysis of the change of the symbiotic phenotype was analysed by transcriptomics. The differentially expressed genes identified are mostly involved in metabolic pathways. However, the transcriptional response also includes genes belonging to other functional categories such as signalling, stress response and transcriptional regulation. Some of these genes show a molecular identity and a regulation profile, that suggest a role as possible molecular links between the N-dependent plant response and the nodule organogenesis program.

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

Highlight Multiple stress signalling pathways regulate LHCX family gene expression in the diatom Phaeodactylum tricornutum to attune acclimation responses efficiently in highly variable ocean environments.

The diatom molecular toolkit to handle nitrogen uptake

Nutrient concentrations in the oceans display significant temporal and spatial variability, which strongly affects growth, distribution and survival of phytoplankton. Nitrogen (N) in particular is often considered a limiting resource for prominent marine microalgae, such as diatoms. Diatoms possess a suite of N-related transporters and enzymes and utilize a variety of inorganic (e.g., nitrate, NO3(-); ammonium, NH4(+)) and organic (e.g., urea; amino acids) N sources for growth. However, the molecular mechanisms allowing diatoms to cope efficiently with N oscillations by controlling uptake capacities and signaling pathways involved in the perception of external and internal clues remain largely unknown. Data reported in the literature suggest that the regulation and the characteristic of the genes, and their products, involved in N metabolism are often diatom-specific, which correlates with the peculiar physiology of these organisms for what N utilization concerns. Our study reveals that diatoms host a larger suite of N transporters than one would expected for a unicellular organism, which may warrant flexible responses to variable conditions, possibly also correlated to the phases of life cycle of the cells. All this makes N transporters a crucial key to reveal the balance between proximate and ultimate factors in diatom life.

Improved procedures for in vitro regeneration and for phenotypic analysis in the model legume Lotus japonicus

As a prerequisite for the development of an efficient gene transfer methodology, the possibility of inducing direct somatic embryogenesis in Lotus japonicus (Regel) K. Larsen explants was investigated. Petiole bases, cotyledons, hypocotyls and stem segments were cultivated in the presence of different amounts of benzylaminopurine (BAP) and / or thidiazuron (TDZ). Regeneration was achieved differentially in the different explants and a higher efficiency of shoot formation was obtained with TDZ. By maintaining the same TDZ regime a second cycle of morphogenesis was achieved and the histological analysis of these structures indicated unambiguously their somatic embryogenic nature. Thidiazuron was also tested as an agent to improve the kinetics of shoot formation in a Lotus japonicus transformation-regeneration procedure based on indirect organogenesis. A very significant, highly reproducible, increase in the rate of the shoot formation was observed in independent transformation experiments. We also present an extensive analysis of the feasibility and reproducibility of an in vitro procedure, which can be very useful for the screening of symbiotic phenotypes in transgenic Lotus plants and for the analysis of the cascade of molecular and cytological events occurring soon after Mesorhizobium loti infection.