Expression and RNA binding properties of the chloroplast ribosomal protein S1 from Chlamydomonas reinhardtii

The gene encoding the chloroplast ribosomal protein S1 from Chlamydomonas reinhardtii, CreS1, was cloned and the RNA binding properties and the expression patterns were studied. Gel-shift analysis revealed that CreS1 binds AU-rich 5′-untranslated regions (5′-UTR) of chloroplast mRNAs with higher affinity than the corresponding sequence of a GC-rich nuclear transcript. The binding affinity of CreS1 for a mutant form of the psbD 5′-UTR with a deletion of a U-rich stretch that is required for translation decreases 4-fold as compared to the wild-type 5′-UTR. Our results suggest that CreS1 protein interacts with U-rich sequences. Most of CreS1 is bound to high-molecular-weight complexes which co-migrate with the 30S small ribosomal subunit, and only a small fraction of CreS1 exists in its free form. CreS1 is localized mainly to the chloroplast stroma albeit a significant fraction is associated with chloroplast membranes. The results suggest that most of CreS1 is associated with the 30S ribosomal subunit throughout the translation process. Upon a shift of cells from the dark to the light, the mRNA levels of CreS1 and Psrp-7, both components of the 30S ribosomal subunit, increase transiently and return to the dark levels after 8h. However, during this dark-to-light transition the levels of CreS1 and of other components of the 30S subunit remain the same suggesting that either protein synthesis or degradation is regulated. The possible implications of these findings are discusse

Rhythms and Clocks in Marine Organisms

The regular movements of waves and tides are obvious representations of the oceans’ rhythmicity. But the rhythms of marine life span across ecological niches and timescales, including short (in the range of hours) and long (in the range of days and months) periods. These rhythms regulate the physiology and behavior of individuals, as well as their interactions with each other and with the environment. This review highlights examples of rhythmicity in marine animals and algae that represent important groups of marine life across different habitats. The examples cover ecologically highly relevant species and a growing number of laboratory model systems that are used to disentangle key mechanistic principles. The review introduces fundamental concepts of chronobiology, such as the distinction between rhythmic and endogenous oscillator–driven processes. It also addresses the relevance of studying diverse rhythms and oscillators, as well as their interconnection, for making better predictions of how species will respond to environmental perturbations, including climate change. As the review aims to address scientists from the diverse fields of marine biology, ecology, and molecular chronobiology, all of which have their own scientific terms, we provide definitions of key terms throughout the article

Effect of Cromoglycate on Gas Changes, During Bronchial Challenge by UNCDW in Children with Asthma

Eighteen asthmatic children were challenged with ultrasonically nebulized cold distilled water (UNCDW). Blood gas composition was monitored transcutaneously (tcpO2 and tcpCO2) during and after the challenge. Assuming as basal the response to this UNCDW test, nine children (Group A) were then chosen at random to inhale cromoglycate by aerosol delivery for 8 days. Nine children (Group B), acting as a control, inhaled saline for 8 days. At the end of this therapy, each child repeated the UNCDW test. Statistical analysis with t-test for paired data was used to compare the results of each child to both tests. Mean basal tcpO2 and tcpCO2 were all within the expected normal range. In all children, both mean tcpO2 and tcpCO2 were reduced during and after UNCDW inhalation. Mean tcpCO2 values during the challenge were significantly (p < 0.001) lower than the corresponding steady state 2 rain after the UNCDW challenge, with a mean drop of −7% (2.1 S.D.). Mean tcpO2 values remained significantly decreased (p < 0.001) from the fifth mitt of the UNCDW challenge to the end of the observation period, with a mean drop of −20% (15.5 S.D.). After treatment with cromoglycate (Group A), the mean tcpCO2 values during UNCDW did not change significantly from those ofsteady state conditions: −0.8% (0.5 S.D.); whereas mean tcpO2 values decreased by −4% (4.9 S.D.). The control children treated with saline (Group B) showed mean tcpCO2 and tcpO2 values which were significantly different (p < 0.001) from those of the steady state conditions: mean drop of tcpCO2, −6% (4.2 S.D.); mean drop of tcpO2, −20% (4.7 S.D.). In conclusion, it emerges that: UNCDW induces nonspecific broncho-constriction in asthmatic children with a typical drop of tcpCO2 and tcpO2; the treatment with cromoglycate normalizes the time course of tcpCO2 (hyper-reactivity) and reduces dramatically the drop of tcpO2 time course (hyper-responsivity) during and after the UNCDW test

The circadian clock in the diatom <i>Phaeodactylum tricornutum</i>

International audienceno abstrac

Diatom molecular research comes of age: Model species for studying phytoplankton biology and diversity

Diatoms are the world’s most diverse group of algae, comprising at least 100,000 species. Contributing;20% of annual global carbon fixation, they underpin major aquatic food webs and drive global biogeochemical cycles. Over the past two decades, Thalassiosira pseudonana and Phaeodactylum tricornutum have become the most important model systems for diatom molecular research, ranging from cell biology to ecophysiology, due to their rapid growth rates, small genomes, and the cumulative wealth of associated genetic resources. To explore the evolutionary divergence of diatoms, additional model species are emerging, such as Fragilariopsis cylindrus and Pseudo-nitzschia multistriata. Here, we describe how functional genomics and reverse genetics have contributed to our understanding of this important class of microalgae in the context of evolution, cell biology, and metabolic adaptations. Our review will also highlight promising areas of investigation into the diversity of these photosynthetic organisms, including the discovery of new molecular pathways governing the life of secondary plastid-bearing organisms in aquatic environments

AUREOCHROME1a-mediated induction of the diatom-specific cyclin dsCYC2 controls the onset of cell division in diatoms (Phaeodactylum tricornutum)

Cell division in photosynthetic organisms is tightly regulated by light. Although the light dependency of the onset of the cell cycle has been well characterized in various phototrophs, little is known about the cellular signaling cascades connecting light perception to cell cycle activation and progression. Here, we demonstrate that diatom-specific cyclin 2 (dsCYC2) in Phaeodactylum tricornutum displays a transcriptional peak within 15 min after light exposure, long before the onset of cell division. The product of dsCYC2 binds to the cyclin-dependent kinase CDKA1 and can complement G1 cyclin-deficient yeast. Consistent with the role of dsCYC2 in controlling a G1-to-S light-dependent cell cycle checkpoint, dsCYC2 silencing decreases the rate of cell division in diatoms exposed to light-dark cycles but not to constant light. Transcriptional induction of dsCYC2 is triggered by blue light in a fluence rate-dependent manner. Consistent with this, dsCYC2 is a transcriptional target of the blue light sensor AUREOCHROME1a, which functions synergistically with the basic leucine zipper (bZIP) transcription factor bZIP10 to induce dsCYC2 transcription. The functional characterization of a cyclin whose transcription is controlled by light and whose activity connects light signaling to cell cycle progression contributes significantly to our understanding of the molecular mechanisms underlying light-dependent cell cycle onset in diatoms

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

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