Far-red light enrichment affects gene expression and architecture as well as growth and photosynthesis in rice.

Plants use light as a resource and signal. Photons within the 400-700 nm waveband are considered photosynthetically active. Far-red photons (FR, 700-800 nm) are used by plants to detect nearby vegetation and elicit the shade avoidance syndrome. In addition, FR photons have also been shown to contribute to photosynthesis, but knowledge about these dual effects remains scarce. Here, we study shoot-architectural and photosynthetic responses to supplemental FR light during the photoperiod in several rice varieties. We observed that FR enrichment only mildly affected the rice transcriptome and shoot architecture as compared to established model species, whereas leaf formation, tillering and biomass accumulation were clearly promoted. Consistent with this growth promotion, we found that CO2-fixation in supplemental FR was strongly enhanced, especially in plants acclimated to FR-enriched conditions as compared to control conditions. This growth promotion dominates the effects of FR photons on shoot development and architecture. When substituting FR enrichment with an end-of-day FR pulse, this prevented photosynthesis-promoting effects and elicited shade avoidance responses. We conclude that FR photons can have a dual role, where effects depend on the environmental context: in addition to being an environmental signal, they are also a potent source of harvestable energy.

Plant enhancers exhibit both cooperative and additive interactions among their functional elements.

Enhancers are cis-regulatory elements that shape gene expression in response to numerous developmental and environmental cues. In animals, several models have been proposed to explain how enhancers integrate the activity of multiple transcription factors. However, it remains largely unclear how plant enhancers integrate transcription factor activity. Here, we use Plant STARR-seq to characterize three light-responsive plant enhancers-AB80, Cab-1, and rbcS-E9-derived from genes associated with photosynthesis. Saturation mutagenesis revealed mutations, many of which clustered in short regions, that strongly reduced enhancer activity in the light, in the dark or in both conditions. When tested in the light, these mutation-sensitive regions did not function on their own; rather, cooperative interactions with other such regions were required for full activity. Epistatic interactions occurred between mutations in adjacent mutation-sensitive regions, and the spacing and order of mutation-sensitive regions in synthetic enhancers affected enhancer activity. In contrast, when tested in the dark, mutation-sensitive regions acted independently and additively in conferring enhancer activity. Taken together, this work demonstrates that plant enhancers show evidence for both cooperative and additive interactions among their functional elements. This knowledge can be harnessed to design strong, condition-specific synthetic enhancers.

Plant signaling: The sugar-coated story of root growth.

A new study draws attention to photosynthetically produced sucrose as a major shoot-derived and auxin-dependent regulator of root growth and development in plants.

COLD REGULATED GENE 27 and 28 antagonize the transcriptional activity of the RVE8/LNK1/LNK2 circadian complex.

Many molecular and physiological processes in plants occur at a specific time of day. These daily rhythms are coordinated in part by the circadian clock, a timekeeper that uses daylength and temperature to maintain rhythms of ∼24 h in various clock-regulated phenotypes. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover connections within the clock and between clock elements and other pathways, here, we used affinity purification coupled with mass spectrometry (APMS) to identify time-of-day-specific protein interactors of the RVE8-LNK1/LNK2 complex in Arabidopsis (Arabidopsis thaliana). Among the interactors of RVE8/LNK1/LNK2 were COLD-REGULATED GENE 27 (COR27) and COR28, which coprecipitated in an evening-specific manner. In addition to COR27 and COR28, we found an enrichment of temperature-related interactors that led us to establish a previously uncharacterized role for LNK1 and LNK2 in temperature entrainment of the clock. We established that RVE8, LNK1, and either COR27 or COR28 form a tripartite complex in yeast (Saccharomyces cerevisiae) and that the effect of this interaction in planta serves to antagonize transcriptional activation of RVE8 target genes, potentially through mediating RVE8 protein degradation in the evening. Together, these results illustrate how a proteomic approach can be used to identify time-of-day-specific protein interactions. Discovery of the RVE8-LNK-COR protein complex indicates a previously unknown regulatory mechanism for circadian and temperature signaling pathways.

PIF7 controls leaf cell proliferation through an AN3 substitution repression mechanism.

Plants are agile, plastic organisms able to adapt to everchanging circumstances. Responding to far-red (FR) wavelengths from nearby vegetation, shade-intolerant species elicit the adaptive shade-avoidance syndrome (SAS), characterized by elongated petioles, leaf hyponasty, and smaller leaves. We utilized end-of-day FR (EODFR) treatments to interrogate molecular processes that underlie the SAS leaf response. Genetic analysis established that PHYTOCHROME-INTERACTING FACTOR 7 (PIF7) is required for EODFR-mediated constraint of leaf blade cell division, while EODFR messenger RNA sequencing data identified ANGUSTIFOLIA3 (AN3) as a potential PIF7 target. We show that PIF7 can suppress AN3 transcription by directly interacting with and sequestering AN3. We also establish that PIF7 and AN3 impose antagonistic control of gene expression via common cis-acting promoter motifs in several cell-cycle regulator genes. EODFR triggers the molecular substitution of AN3 to PIF7 at G-box/PBE-box promoter regions and a switch from promotion to repression of gene expression.

Phytochrome regulates cellular response plasticity and the basic molecular machinery of leaf development.

Plants are plastic organisms that optimize growth in response to a changing environment. This adaptive capability is regulated by external cues, including light, which provides vital information about the habitat. Phytochrome photoreceptors detect far-red light, indicative of nearby vegetation, and elicit the adaptive shade-avoidance syndrome (SAS), which is critical for plant survival. Plants exhibiting SAS are typically more elongated, with distinctive, small, narrow leaf blades. By applying SAS-inducing end-of-day far-red (EoD FR) treatments at different times during Arabidopsis (Arabidopsis thaliana) leaf 3 development, we have shown that SAS restricts leaf blade size through two distinct cellular strategies. Early SAS induction limits cell division, while later exposure limits cell expansion. This flexible strategy enables phytochromes to maintain control of leaf size through the proliferative and expansion phases of leaf growth. mRNAseq time course data, accessible through a community resource, coupled to a bioinformatics pipeline, identified pathways that underlie these dramatic changes in leaf growth. Phytochrome regulates a suite of major development pathways that control cell division, expansion, and cell fate. Further, phytochromes control cell proliferation through synchronous regulation of the cell cycle, DNA replication, DNA repair, and cytokinesis, and play an important role in sustaining ribosome biogenesis and translation throughout leaf development.

Phytochromes control metabolic flux, and their action at the seedling stage determines adult plant biomass.

Phytochrome photoreceptors are known to regulate plastic growth responses to vegetation shade. However, recent reports also suggest an important role for phytochromes in carbon resource management, metabolism, and growth. Here, we use 13CO2 labelling patterns in multiallele phy mutants to investigate the role of phytochrome in the control of metabolic fluxes. We also combine quantitative data of 13C incorporation into protein and cell wall polymers, gas exchange measurements, and system modelling to investigate why biomass is decreased in adult multiallele phy mutants. Phytochrome influences the synthesis of stress metabolites such as raffinose and proline, and the accumulation of sugars, possibly through regulating vacuolar sugar transport. Remarkably, despite their modified metabolism and vastly altered architecture, growth rates in adult phy mutants resemble those of wild-type plants. Our results point to delayed seedling growth and smaller cotyledon size as the cause of the adult-stage phy mutant biomass defect. Our data signify a role for phytochrome in metabolic stress physiology and carbon partitioning, and illustrate that phytochrome action at the seedling stage sets the trajectory for adult biomass production.

Global transcriptome analysis reveals circadian control of splicing events in Arabidopsis thaliana.

The circadian clock of Arabidopsis thaliana controls many physiological and molecular processes, allowing plants to anticipate daily changes in their environment. However, developing a detailed understanding of how oscillations in mRNA levels are connected to oscillations in co/post-transcriptional processes, such as splicing, has remained a challenge. Here we applied a combined approach using deep transcriptome sequencing and bioinformatics tools to identify novel circadian-regulated genes and splicing events. Using a stringent approach, we identified 300 intron retention, eight exon skipping, 79 alternative 3' splice site usage, 48 alternative 5' splice site usage, and 350 multiple (more than one event type) annotated events under circadian regulation. We also found seven and 721 novel alternative exonic and intronic events. Depletion of the circadian-regulated splicing factor AtSPF30 homologue resulted in the disruption of a subset of clock-controlled splicing events. Altogether, our global circadian RNA-seq coupled with an in silico, event-centred, splicing analysis tool offers a new approach for studying the interplay between the circadian clock and the splicing machinery at a global scale. The identification of many circadian-regulated splicing events broadens our current understanding of the level of control that the circadian clock has over this co/post-transcriptional regulatory layer.

Bacterial Infection Disrupts Clock Gene Expression to Attenuate Immune Responses.

The circadian clock modulates immune responses in plants and animals; however, it is unclear how host-pathogen interactions affect the clock. Here we analyzed clock function in Arabidopsis thaliana mutants with defective immune responses and found that enhanced disease susceptibility 4 (eds4) displays alterations in several circadian rhythms. Mapping by sequencing revealed that EDS4 encodes the ortholog of NUCLEOPORIN 205, a core component of the inner ring of the nuclear pore complex (NPC). Consistent with the idea that the NPC specifically modulates clock function, we found a strong enrichment in core clock genes, as well as an increased nuclear to total mRNA accumulation, among genes that were differentially expressed in eds4 mutants. Interestingly, infection with Pseudomonas syringae in wild-type (WT) plants downregulated the expression of several morning core clock genes as early as 1 h post-infection, including all members of the NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED (LNK) gene family, and this effect was attenuated in eds4. Furthermore, lnk mutants were more susceptible than the WT to P. syringae infection. These results indicate that bacterial infection, acting in part through the NPC, alters core clock gene expression and/or mRNA accumulation in a way that favors bacterial growth and disease susceptibility.

Probiotic Bacillus subtilis Protects against α-Synuclein Aggregation in C. elegans.

Recent discoveries have implicated the gut microbiome in the progression and severity of Parkinson's disease; however, how gut bacteria affect such neurodegenerative disorders remains unclear. Here, we report that the Bacillus subtilis probiotic strain PXN21 inhibits α-synuclein aggregation and clears preformed aggregates in an established Caenorhabditis elegans model of synucleinopathy. This protection is seen in young and aging animals and is partly mediated by DAF-16. Multiple B. subtilis strains trigger the protective effect via both spores and vegetative cells, partly due to a biofilm formation in the gut of the worms and the release of bacterial metabolites. We identify several host metabolic pathways differentially regulated in response to probiotic exposure, including sphingolipid metabolism. We further demonstrate functional roles of the sphingolipid metabolism genes lagr-1, asm-3, and sptl-3 in the anti-aggregation effect. Our findings provide a basis for exploring the disease-modifying potential of B. subtilis as a dietary supplement.

The LNK Gene Family: At the Crossroad between Light Signaling and the Circadian Clock.

Light signaling pathways interact with the circadian clock to help organisms synchronize physiological and developmental processes to periodic environmental cycles. The plant photoreceptors responsible for clock resetting have been characterized, but signaling components that link the photoreceptors to the clock remain to be identified. Members of the family of NIGHT LIGHT⁻INDUCIBLE AND CLOCK-REGULATED (LNK) genes play key roles linking light regulation of gene expression to the control of daily and seasonal rhythms in Arabidopsis thaliana. Particularly, LNK1 and LNK2 were shown to control circadian rhythms, photomorphogenic responses, and photoperiod-dependent flowering time. Here we analyze the role of the four members of the LNK family in Arabidopsis in these processes. We found that depletion of the closely related LNK3 and LNK4 in a lnk1;lnk2 mutant background affects circadian rhythms, but not other clock-regulated processes such as flowering time and seedling photomorphogenesis. Nevertheless, plants defective in all LNK genes (lnkQ quadruple mutants) display developmental alterations that lead to increased rosette size, biomass, and enhanced phototropic responses. Our work indicates that members of the LNK family have both distinctive and partially overlapping functions, and are an essential link to orchestrate light-regulated developmental processes.

Transcriptional and post-transcriptional control of the plant circadian gene regulatory network.

The circadian clock drives rhythms in multiple physiological processes allowing plants to anticipate and adjust to periodic changes in environmental conditions. These physiological rhythms are associated with robust oscillations in the expression of thousands of genes linked to the control of photosynthesis, cell elongation, biotic and abiotic stress responses, developmental processes such as flowering, and the clock itself. Given its pervasive effects on plant physiology, it is not surprising that circadian clock genes have played an important role in the domestication of crop plants and in the improvement of crop productivity. Therefore, identifying the principles governing the dynamics of the circadian gene regulatory network in plants could strongly contribute to further speed up crop improvement. Here we provide an historical as well as a current description of our knowledge of the molecular mechanisms underlying circadian rhythms in plants. This work focuses on the transcriptional and post-transcriptional regulatory layers that control the very core of the circadian clock, and some of its complex interactions with signaling pathways that help synchronize plant growth and development to daily and seasonal changes in the environment. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

Circadian rhythms identified in Caenorhabditis elegans by in vivo long-term monitoring of a bioluminescent reporter.

Circadian rhythms are based on endogenous clocks that allow organisms to adjust their physiology and behavior by entrainment to the solar day and, in turn, to select the optimal times for most biological variables. Diverse model systems-including mice, flies, fungi, plants, and bacteria-have provided important insights into the mechanisms of circadian rhythmicity. However, the general principles that govern the circadian clock of Caenorhabditis elegans have remained largely elusive. Here we report robust molecular circadian rhythms in C elegans recorded with a bioluminescence assay in vivo and demonstrate the main features of the circadian system of the nematode. By constructing a luciferase-based reporter coupled to the promoter of the suppressor of activated let-60 Ras (sur-5) gene, we show in both population and single-nematode assays that C elegans expresses ∼24-h rhythms that can be entrained by light/dark and temperature cycles. We provide evidence that these rhythms are temperature-compensated and can be re-entrained after phase changes of the synchronizing agents. In addition, we demonstrate that light and temperature sensing requires the photoreceptors LITE and GUR-3, and the cyclic nucleotide-gated channel subunit TAX-2. Our results shed light on C elegans circadian biology and demonstrate evolutionarily conserved features in the circadian system of the nematode.

Acute Effects of Light on Alternative Splicing in Light-Grown Plants.

Light modulates plant growth and development to a great extent by regulating gene expression programs. Here, we evaluated the effect of light on alternative splicing (AS) in light-grown Arabidopsis thaliana plants using high-throughput RNA sequencing (RNA-seq). We found that an acute light pulse given in the middle of the night, a treatment that simulates photoperiod lengthening, affected AS events corresponding to 382 genes. Some of these AS events were associated with genes involved in primary metabolism and stress responses, which may help to adjust metabolic and physiological responses to seasonal changes. We also found that several core clock genes showed changes in AS in response to the light treatment, suggesting that light regulation of AS may play a role in clock entrainment. Finally, we found that many light-regulated AS events were associated with genes encoding RNA processing proteins and splicing factors, supporting the idea that light regulates this posttranscriptional regulatory layer through AS regulation of splicing factors. Interestingly, the effect of a red-light pulse on AS of a gene encoding a splicing factor was not impaired in a quintuple phytochrome mutant, providing unequivocal evidence that nonphotosensory photoreceptors control AS in light-grown plants.

Circadian rhythms and post-transcriptional regulation in higher plants.

The circadian clock of plants allows them to cope with daily changes in their environment. This is accomplished by the rhythmic regulation of gene expression, in a process that involves many regulatory steps. One of the key steps involved at the RNA level is post-transcriptional regulation, which ensures a correct control on the different amounts and types of mRNA that will ultimately define the current physiological state of the plant cell. Recent advances in the study of the processes of regulation of pre-mRNA processing, RNA turn-over and surveillance, regulation of translation, function of lncRNAs, biogenesis and function of small RNAs, and the development of bioinformatics tools have helped to vastly expand our understanding of how this regulatory step performs its role. In this work we review the current progress in circadian regulation at the post-transcriptional level research in plants. It is the continuous interaction of all the information flow control post-transcriptional processes that allow a plant to precisely time and predict daily environmental changes.

Potential conservation of circadian clock proteins in the phylum Nematoda as revealed by bioinformatic searches.

Although several circadian rhythms have been described in C. elegans, its molecular clock remains elusive. In this work we employed a novel bioinformatic approach, applying probabilistic methodologies, to search for circadian clock proteins of several of the best studied circadian model organisms of different taxa (Mus musculus, Drosophila melanogaster, Neurospora crassa, Arabidopsis thaliana and Synechoccocus elongatus) in the proteomes of C. elegans and other members of the phylum Nematoda. With this approach we found that the Nematoda contain proteins most related to the core and accessory proteins of the insect and mammalian clocks, which provide new insights into the nematode clock and the evolution of the circadian system.

Daily variation in melatonin synthesis and arylalkylamine N-acetyltransferase activity in the nematode Caenorhabditis elegans.

Melatonin influences circadian rhythms and seasonal behavioral changes in vertebrates; it is synthesized from serotonin by N-acetylation by arylalkylamine N-acetyltransferase (AA-NAT) and O-methylation by N-acetylserotonin methyltransferase. However, its physiology and function in invertebrate models are less understood. In this work, we studied daily variations in melatonin synthesis and AA-NAT activity in the nematode Caenorhabditis elegans. Under light-dark conditions (LD), a rhythmic pattern of melatonin levels was observed, with higher levels toward the middle of the night, peaking at zeitgeber time (ZT) 18, and with a minimum value around ZT0-6. AA-NAT activity showed a diurnal and circadian fluctuation with higher levels of activity during the early night, both under LD and constant darkness conditions. A peak was found around ZT12 and circadian time (CT) 12. In addition, we investigated whether this nocturnal AA-NAT activity is inhibited by light. Our results show that both white and blue light pulses significantly inhibited AA-NAT activity at ZT18. This work demonstrates the daily fluctuation of melatonin synthesis and AA-NAT activity in the adult nematode C. elegans. In summary, this study takes additional advantage of an extremely useful invertebrate model system, which has only recently been exploited for circadian studies.

Circadian rhythms in metabolic variables in Caenorhabditis elegans.

Circadian rhythms govern a wide variety of physiological and metabolic functions in most organisms through neural networks, hormones and gene expression. In this work, we studied the circadian variation in metabolic variables of adult C. elegans such as food consumption, pharyngeal contractions, defecation and oxygen consumption. Feeding behavior was clearly rhythmic under LD conditions, with a non-significant trend under DD conditions. In addition, a daily and circadian variation in muscle contraction of the pharynx was observed. Oxygen consumption also showed a circadian fluctuation with a maximum in the middle of the night (a peak was found around ZT18/CT18). Furthermore, defecation behavior also showed a daily variation in the N2 strain (wild type). This work demonstrates that in the adult nematode C. elegans metabolic variables vary daily. In summary, our results will allow us to take full advantage of this widely used animal model (including research in genetics, ageing and developmental biology) for studies in Chronobiology.

Circadian variation in Pseudomonas fluorescens (CHA0)-mediated paralysis of Caenorhabditis elegans.

Abiotic and biotic environmental stressors play a key role in the ecophysiology of most organisms. As the presence and activity of stress-inducing agents vary along the day, organisms that are able to predict these periodic changes are better fit to survive. Caenorhabditis elegans, a soil-dwelling nematode, is subjected to daily changes in its natural environment, and its tolerance to osmotic and oxidative stress varies along the day. Pseudomonas fluorescens strain CHA0 is a soil bacterium that produces a set of secondary metabolites that antagonize phytopathogenic fungi and therefore promote healthy growth of several plant species. Here we show that strain CHA0 is able to affect C. elegans either under growth limiting conditions (i.e., slow-killing) or by rapid paralysis in nutrient replete conditions (fast-killing). Both types of toxicity require the post-transcriptional Gac/Rsm regulatory cascade, and the fast paralytic killing depends strongly on hydrogen cyanide production. The response observed in C. elegans nematodes to fast paralytic killing varies along the day and its sensitivity is higher during the night, at Zeitgeber Time (ZT) 12 (lights off). This behavior correlates well with HCN tolerance, which is higher during the day, at ZT0 (lights on). The innate immune response to P. fluorescens CHA0 might depend on the stress response pathway of C. elegans. The fact that the tolerance varies daily gives further proof of an underlying clock that governs cyclic behavior in C. elegans.