Timely symbiosis: circadian control of legume-rhizobia symbiosis.

Legumes house nitrogen-fixing endosymbiotic rhizobia in specialised polyploid cells within root nodules. This results in a mutualistic relationship whereby the plant host receives fixed nitrogen from the bacteria in exchange for dicarboxylic acids. This plant-microbe interaction requires the regulation of multiple metabolic and physiological processes in both the host and symbiont in order to achieve highly efficient symbiosis. Recent studies have showed that the success of symbiosis is influenced by the circadian clock of the plant host. Medicago and soybean plants with altered clock mechanisms showed compromised nodulation and reduced plant growth. Furthermore, transcriptomic analyses revealed that multiple genes with key roles in recruitment of rhizobia to plant roots, infection and nodule development were under circadian control, suggesting that appropriate timing of expression of these genes may be important for nodulation. There is also evidence for rhythmic gene expression of key nitrogen fixation genes in the rhizobium symbiont, and temporal coordination between nitrogen fixation in the bacterial symbiont and nitrogen assimilation in the plant host may be important for successful symbiosis. Understanding of how circadian regulation impacts on nodule establishment and function will identify key plant-rhizobial connections and regulators that could be targeted to increase the efficiency of this relationship.

Prediction of photoperiodic regulators from quantitative gene circuit models.

Photoperiod sensors allow physiological adaptation to the changing seasons. The prevalent hypothesis is that day length perception is mediated through coupling of an endogenous rhythm with an external light signal. Sufficient molecular data are available to test this quantitatively in plants, though not yet in mammals. In Arabidopsis, the clock-regulated genes CONSTANS (CO) and FLAVIN, KELCH, F-BOX (FKF1) and their light-sensitive proteins are thought to form an external coincidence sensor. Here, we model the integration of light and timing information by CO, its target gene FLOWERING LOCUS T (FT), and the circadian clock. Among other predictions, our models show that FKF1 activates FT. We demonstrate experimentally that this effect is independent of the known activation of CO by FKF1, thus we locate a major, novel controller of photoperiodism. External coincidence is part of a complex photoperiod sensor: modeling makes this complexity explicit and may thus contribute to crop improvement.

Genetic and physiological responses to light quality in a deep ocean ecotype of Ostreococcus, an ecologically important photosynthetic picoeukaryote.

Phytoplankton are exposed to dramatic variations in light quality when cells are carried by upwelling or downwelling currents or encounter sediment. We investigated the potential impact of light quality changes in Ostreococcus, a key marine photosynthetic picoeukaryote, by analysing changes in its transcriptome, pigment content, and photophysiology after acclimation to monochromatic red, green, or blue light. The clade B species RCC809, isolated from the deep euphotic zone of the tropical Atlantic Ocean, responded to blue light by accelerating cell division at the expense of storage reserves and by increasing the relative level of blue-light-absorbing pigments. It responded to red and green light by increasing its potential for photoprotection. In contrast, the clade A species OTTH0595, which originated from a shallow water environment, showed no difference in photosynthetic properties and minor differences in carotenoid contents between light qualities. This was associated with the loss of candidate light-quality responsive promoter motifs identified in RCC809 genes. These results demonstrate that light quality can have a major influence on the physiology of eukaryotic phytoplankton and suggest that different light quality environments can drive selection for diverse patterns of responsiveness and environmental niche partitioning.

Circadian rhythms in the plant host influence rhythmicity of rhizosphere microbiota.

BACKGROUND: Recent studies demonstrated that microbiota inhabiting the plant rhizosphere exhibit diel changes in abundance. To investigate the impact of plant circadian rhythms on bacterial and fungal rhythms in the rhizosphere, we analysed temporal changes in fungal and bacterial communities in the rhizosphere of Arabidopsis plants overexpressing or lacking function of the circadian clock gene LATE ELONGATED HYPOCOTYL (LHY). RESULTS: Under diel light-dark cycles, the knock-out mutant lhy-11 and the gain-of-function mutant lhy-ox both exhibited gene expression rhythms with altered timing and amplitude compared to wild-type plants. Distinct sets of bacteria and fungi were found to display rhythmic changes in abundance in the rhizosphere of both of these mutants, suggesting that abnormal patterns of rhythmicity in the plant host caused temporal reprogramming of the rhizosphere microbiome. This was associated with changes in microbial community structure, including changes in the abundance of fungal guilds known to impact on plant health. Under constant environmental conditions, microbial rhythmicity persisted in the rhizosphere of wild-type plants, indicating control by a circadian oscillator. In contrast, loss of rhythmicity in lhy-ox plants was associated with disrupted rhythms for the majority of rhizosphere microbiota. CONCLUSIONS: These results show that aberrant function of the plant circadian clock is associated with altered rhythmicity of rhizosphere bacteria and fungi. In the long term, this leads to changes in composition of the rhizosphere microbiome, with potential consequences for plant health. Further research will be required to understand the functional implications of these changes and how they impact on plant health and productivity.

Plant circadian clock control of Medicago truncatula nodulation via regulation of nodule cysteine-rich peptides.

Legumes house nitrogen-fixing endosymbiotic rhizobia in specialized polyploid cells within root nodules, which undergo tightly regulated metabolic activity. By carrying out expression analysis of transcripts over time in Medicago truncatula nodules, we found that the circadian clock enables coordinated control of metabolic and regulatory processes linked to nitrogen fixation. This involves the circadian clock-associated transcription factor LATE ELONGATED HYPOCOTYL (LHY), with lhy mutants being affected in nodulation. Rhythmic transcripts in root nodules include a subset of nodule-specific cysteine-rich peptides (NCRs) that have the LHY-bound conserved evening element in their promoters. Until now, studies have suggested that NCRs act to regulate bacteroid differentiation and keep the rhizobial population in check. However, these conclusions came from the study of a few members of this very large gene family that has complex diversified spatio-temporal expression. We suggest that rhythmic expression of NCRs may be important for temporal coordination of bacterial activity with the rhythms of the plant host, in order to ensure optimal symbiosis.

[Once a caregiver, always a caregiver]. / Soignant un jour, soignant toujours.

March 2020, COVID-19 pandemic. In France, a state of health emergency is declared by the authorities; training managers are called in as reinforcements. This return to the field in the context of a health crisis has made it possible to re-examine practices and the place of the manager within the health system.

Circadian control of abscisic acid biosynthesis and signalling pathways revealed by genome-wide analysis of LHY binding targets.

The LATE ELONGATED HYPOCOTYL (LHY) transcription factor functions as part of the oscillatory mechanism of the Arabidopsis circadian clock. This paper reports the genome-wide analysis of its binding targets and reveals a role in the control of abscisic acid (ABA) biosynthesis and downstream responses. LHY directly repressed expression of 9-cis-epoxycarotenoid dioxygenase enzymes, which catalyse the rate-limiting step of ABA biosynthesis. This suggested a mechanism for the circadian control of ABA accumulation in wild-type plants. Consistent with this hypothesis, ABA accumulated rhythmically in wild-type plants, peaking in the evening. LHY-overexpressing plants had reduced levels of ABA under drought stress, whereas loss-of-function mutants exhibited an altered rhythm of ABA accumulation. LHY also bound the promoter of multiple components of ABA signalling pathways, suggesting that it may also act to regulate responses downstream of the hormone. LHY promoted expression of ABA-responsive genes responsible for increased tolerance to drought and osmotic stress but alleviated the inhibitory effect of ABA on seed germination and plant growth. This study reveals a complex interaction between the circadian clock and ABA pathways, which is likely to make an important contribution to plant performance under drought and osmotic stress conditions.

Jasmonate signalling drives time-of-day differences in susceptibility of Arabidopsis to the fungal pathogen Botrytis cinerea.

The circadian clock, an internal time-keeping mechanism, allows plants to anticipate regular changes in the environment, such as light and dark, and biotic challenges such as pathogens and herbivores. Here, we demonstrate that the plant circadian clock influences susceptibility to the necrotrophic fungal pathogen, Botrytis cinerea. Arabidopsis plants show differential susceptibility to B. cinerea depending on the time of day of inoculation. Decreased susceptibility after inoculation at dawn compared with night persists under constant light conditions and is disrupted in dysfunctional clock mutants, demonstrating the role of the plant clock in driving time-of-day susceptibility to B. cinerea. The decreased susceptibility to B. cinerea following inoculation at subjective dawn was associated with faster transcriptional reprogramming of the defence response with gating of infection-responsive genes apparent. Direct target genes of core clock regulators were enriched among the transcription factors that responded more rapidly to infection at subjective dawn than subjective night, suggesting an influence of the clock on the defence-signalling network. In addition, jasmonate signalling plays a crucial role in the rhythmic susceptibility of Arabidopsis to B. cinerea with the enhanced susceptibility to this pathogen at subjective night lost in a jaz6 mutant.

Emerging design principles in the Arabidopsis circadian clock.

Recent experimental advances have enabled the identification of direct regulatory targets for transcription factors. Application of these techniques to the circadian regulatory network in Arabidopsis has uncovered a number of discrepancies within established models as well as novel regulatory interactions. This review integrates these new findings and discusses the functional implications of the revised transcriptional network for the oscillatory mechanism of the clock.

Efficient gene targeting and removal of foreign DNA by homologous recombination in the picoeukaryote Ostreococcus.

With fewer than 8000 genes and a minimalist cellular organization, the green picoalga Ostreococcus tauri is one of the simplest photosynthetic eukaryotes. Ostreococcus tauri contains many plant-specific genes but exhibits a very low gene redundancy. The haploid genome is extremely dense with few repeated sequences and rare transposons. Thanks to the implementation of genetic transformation and vectors for inducible overexpression/knockdown this picoeukaryotic alga has emerged in recent years as a model organism for functional genomics analyses and systems biology. Here we report the development of an efficient gene targeting technique which we use to knock out the nitrate reductase and ferritin genes and to knock in a luciferase reporter in frame to the ferritin native protein. Furthermore, we show that the frequency of insertion by homologous recombination is greatly enhanced when the transgene is designed to replace an existing genomic insertion. We propose that a natural mechanism based on homologous recombination may operate to remove inserted DNA sequences from the genome.

An improved genome of the model marine alga Ostreococcus tauri unfolds by assessing Illumina de novo assemblies.

BACKGROUND: Cost effective next generation sequencing technologies now enable the production of genomic datasets for many novel planktonic eukaryotes, representing an understudied reservoir of genetic diversity. O. tauri is the smallest free-living photosynthetic eukaryote known to date, a coccoid green alga that was first isolated in 1995 in a lagoon by the Mediterranean sea. Its simple features, ease of culture and the sequencing of its 13 Mb haploid nuclear genome have promoted this microalga as a new model organism for cell biology. Here, we investigated the quality of genome assemblies of Illumina GAIIx 75 bp paired-end reads from Ostreococcus tauri, thereby also improving the existing assembly and showing the genome to be stably maintained in culture. RESULTS: The 3 assemblers used, ABySS, CLCBio and Velvet, produced 95% complete genomes in 1402 to 2080 scaffolds with a very low rate of misassembly. Reciprocally, these assemblies improved the original genome assembly by filling in 930 gaps. Combined with additional analysis of raw reads and PCR sequencing effort, 1194 gaps have been solved in total adding up to 460 kb of sequence. Mapping of RNAseq Illumina data on this updated genome led to a twofold reduction in the proportion of multi-exon protein coding genes, representing 19% of the total 7699 protein coding genes. The comparison of the DNA extracted in 2001 and 2009 revealed the fixation of 8 single nucleotide substitutions and 2 deletions during the approximately 6000 generations in the lab. The deletions either knocked out or truncated two predicted transmembrane proteins, including a glutamate-receptor like gene. CONCLUSION: High coverage (>80 fold) paired-end Illumina sequencing enables a high quality 95% complete genome assembly of a compact ~13 Mb haploid eukaryote. This genome sequence has remained stable for 6000 generations of lab culture.

Evolutionarily conserved regulatory motifs in the promoter of the Arabidopsis clock gene LATE ELONGATED HYPOCOTYL.

The transcriptional regulation of the LATE ELONGATED HYPOCOTYL (LHY) gene is key to the structure of the circadian oscillator, integrating information from multiple regulatory pathways. We identified a minimal region of the LHY promoter that was sufficient for rhythmic expression. Another upstream sequence was also required for appropriate waveform of transcription and for maximum amplitude of oscillations under both diurnal and free-running conditions. We showed that two classes of protein complexes interact with a G-box and with novel 5A motifs; mutation of these sites reduced the amplitude of oscillation and broadened the peak of expression. A genome-wide bioinformatic analysis showed that these sites were enriched in phase-specific clusters of rhythmically expressed genes. Comparative genomic analyses showed that these motifs were conserved in orthologous promoters from several species. A position-specific scoring matrix for the 5A sites suggested similarity to CArG boxes, which are recognized by MADS box transcription factors. In support of this, the FLOWERING LOCUS C (FLC) protein was shown to interact with the LHY promoter in planta. This suggests a mechanism by which FLC might affect circadian period.

Chromatin Immunoprecipitation Protocol for Circadian Clock Proteins.

Chromatin immunoprecipitation, or ChIP, is a powerful experimental technique for probing protein-DNA interactions in vivo. This assay can be used to investigate the association of a protein of interest with specific target loci. Alternatively, it can be combined with high-throughput sequencing technology to identify genome-wide binding sites. Here, we describe a ChIP protocol that was optimized for low-abundance transcription factors in Arabidopsis, and provide guidance on how to adapt it for other types of plants and proteins.

Evolutionary analysis of regulatory sequences (EARS) in plants.

Identification of regulatory sequences within non-coding regions of DNA is an essential step towards elucidation of gene networks. This approach constitutes a major challenge, however, as only a very small fraction of non-coding DNA is thought to contribute to gene regulation. The mapping of regulatory regions traditionally involves the laborious construction of promoter deletion series which are then fused to reporter genes and assayed in transgenic organisms. Bioinformatic methods can be used to scan sequences for matches for known regulatory motifs, however these methods are currently hampered by the relatively small amount of such motifs and by a high false-discovery rate. Here, we demonstrate a robust and highly sensitive, in silico method to identify evolutionarily conserved regions within non-coding DNA. Sequence conservation within these regions is taken as evidence for evolutionary pressure against mutations, which is suggestive of functional importance. We test this method on a small set of well characterised promoters, and show that it successfully identifies known regulatory regions. We further show that these evolutionarily conserved sequences contain clusters of transcription binding sites, often described as regulatory modules. A version of the tool optimised for the analysis of plant promoters is available online at http://wsbc.warwick.ac.uk/ears/main.php.

LHY and CCA1 are partially redundant genes required to maintain circadian rhythms in Arabidopsis.

Several genes are known to regulate circadian rhythms in Arabidopsis, but the identity of the central oscillator has not been established. LHY and CCA1 are related MYB-like transcription factors proposed to be closely involved. Here we demonstrate that, as shown previously for CCA1, inactivation of LHY shortens the period of circadian rhythms in gene expression and leaf movements. By constructing lhy cca1-1 double mutants, we show that LHY and CCA1 are partially redundant and essential for the maintenance of circadian rhythms in constant light. Under light/dark cycles the lhy cca1-1 plants show dramatically earlier phases of expression of GI and TOC1, genes associated with the generation of circadian rhythms and the promotion of LHY and CCA1 expression. We conclude that LHY and CCA1 appear to be negative regulatory elements required for central oscillator function.

Isoform switching facilitates period control in the Neurospora crassa circadian clock.

A striking and defining feature of circadian clocks is the small variation in period over a physiological range of temperatures. This is referred to as temperature compensation, although recent work has suggested that the variation observed is a specific, adaptive control of period. Moreover, given that many biological rate constants have a Q(10) of around 2, it is remarkable that such clocks remain rhythmic under significant temperature changes. We introduce a new mathematical model for the Neurospora crassa circadian network incorporating experimental work showing that temperature alters the balance of translation between a short and long form of the FREQUENCY (FRQ) protein. This is used to discuss period control and functionality for the Neurospora system. The model reproduces a broad range of key experimental data on temperature dependence and rhythmicity, both in wild-type and mutant strains. We present a simple mechanism utilising the presence of the FRQ isoforms (isoform switching) by which period control could have evolved, and argue that this regulatory structure may also increase the temperature range where the clock is robustly rhythmic.

Circadian oscillations of cytosolic free calcium regulate the Arabidopsis circadian clock.

In the last decade, the view of circadian oscillators has expanded from transcriptional feedback to incorporate post-transcriptional, post-translational, metabolic processes and ionic signalling. In plants and animals, there are circadian oscillations in the concentration of cytosolic free Ca2+ ([Ca2+]cyt), though their purpose has not been fully characterized. We investigated whether circadian oscillations of [Ca2+]cyt regulate the circadian oscillator of Arabidopsis thaliana. We report that in Arabidopsis, [Ca2+]cyt circadian oscillations can regulate circadian clock function through the Ca2+-dependent action of CALMODULIN-LIKE24 (CML24). Genetic analyses demonstrate a linkage between CML24 and the circadian oscillator, through pathways involving the circadian oscillator gene TIMING OF CAB2 EXPRESSION1 (TOC1).

ELF3: a circadian safeguard to buffer effects of light.

The EARLY FLOWERING 3 (ELF3) gene of Arabidopsis regulates plant morphology, flowering time and circadian rhythms. ELF3 was proposed to function as a modulator of light signal transduction downstream of phytochromes, and, perhaps, other photoreceptors. Recent work indicates that ELF3 encodes a novel nuclear protein that is expressed rhythmically and interacts with phytochrome B. How ELF3 mediates the circadian gating of light responses and regulates light input to the clock is the subject of discussion.

Circadian regulation of abiotic stress tolerance in plants.

Extremes of temperatures, drought and salinity cause widespread crop losses throughout the world and impose severe limitations on the amount of land that can be used for agricultural purposes. Hence, there is an urgent need to develop crops that perform better under such abiotic stress conditions. Here, we discuss intriguing, recent evidence that circadian clock contributes to plants' ability to tolerate different types of environmental stress, and to acclimate to them. The clock controls expression of a large fraction of abiotic stress-responsive genes, as well as biosynthesis and signaling downstream of stress response hormones. Conversely, abiotic stress results in altered expression and differential splicing of the clock genes, leading to altered oscillations of downstream stress-response pathways. We propose a range of mechanisms by which this intimate coupling between the circadian clock and environmental stress-response pathways may contribute to plant growth and survival under abiotic stress.

Revised Morning Loops of the Arabidopsis Circadian Clock Based on Analyses of Direct Regulatory Interactions.

The network structure of the plant circadian clock is complex and direct regulatory interactions between individual components have proven particularly difficult to predict from genetic analyses. Here, we systematically investigate in vivo binding interactions between the morning-specific transcription factor, LATE ELONGATED HYPOCOTYL (LHY) and the promoters of other components of the network. We then demonstrate the functionality of these interactions by testing the responsiveness of the target gene to an ethanol-induced change in expression level of the LHY protein. We uncover novel, negative autoregulatory feedback loops from LHY and the closely related CIRCADIAN CLOCK ASSOCIATED-1 (CCA1) onto their own and each other's expression. Furthermore we show that LHY acts as a repressor of all other clock components, including PSEUDO-RESPONSE REGULATORs (PRRs) 9 and 7, which were previously thought to be positive regulatory targets. These experimental results lead to a substantial revision of the morning loops of the clock.