TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth.

Plant photoperiodic growth is coordinated by interactions between circadian clock and light signaling networks. How post-translational modifications of clock proteins affect these interactions to mediate rhythmic growth remains unclear. Here, we identify five phosphorylation sites in the Arabidopsis core clock protein TIMING OF CAB EXPRESSION 1 (TOC1) which when mutated to alanine eliminate detectable phosphorylation. The TOC1 phospho-mutant fails to fully rescue the clock, growth, and flowering phenotypes of the toc1 mutant. Further, the TOC1 phospho-mutant shows advanced phase, a faster degradation rate, reduced interactions with PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) and HISTONE DEACETYLASE 15 (HDA15), and poor binding at pre-dawn hypocotyl growth-related genes (PHGs), leading to a net de-repression of hypocotyl growth. NUCLEAR FACTOR Y subunits B and C (NF-YB/C) stabilize TOC1 at target promoters, and this novel trimeric complex (NF-TOC1) acts as a transcriptional co-repressor with HDA15 to inhibit PIF-mediated hypocotyl elongation. Collectively, we identify a molecular mechanism suggesting how phosphorylation of TOC1 alters its phase, stability, and physical interactions with co-regulators to precisely phase PHG expression to control photoperiodic hypocotyl growth.

The Na+/H+ antiporter SALT OVERLY SENSITIVE 1 regulates salt compensation of circadian rhythms by stabilizing GIGANTEA in Arabidopsis.

The circadian clock is a timekeeping, homeostatic system that temporally coordinates all major cellular processes. The function of the circadian clock is compensated in the face of variable environmental conditions ranging from normal to stress-inducing conditions. Salinity is a critical environmental factor affecting plant growth, and plants have evolved the SALT OVERLY SENSITIVE (SOS) pathway to acquire halotolerance. However, the regulatory systems for clock compensation under salinity are unclear. Here, we show that the plasma membrane Na+/H+ antiporter SOS1 specifically functions as a salt-specific circadian clock regulator via GIGANTEA (GI) in Arabidopsis thaliana. SOS1 directly interacts with GI in a salt-dependent manner and stabilizes this protein to sustain a proper clock period under salinity conditions. SOS1 function in circadian clock regulation requires the salt-mediated secondary messengers cytosolic free calcium and reactive oxygen species, pointing to a distinct regulatory role for SOS1 in addition to its function as a transporter to maintain Na+ homeostasis. Our results demonstrate that SOS1 maintains homeostasis of the salt response under high or daily fluctuating salt levels. These findings highlight the genetic capacity of the circadian clock to maintain timekeeping activity over a broad range of salinity levels.

NUA positively regulates plant immunity by coordination with ESD4 to deSUMOylate TPR1 in Arabidopsis.

Nuclear pore complex (NPC) is composed of multiple nucleoporins (Nups). A plethora of studies have highlighted the significance of NPC in plant immunity. However, the specific roles of individual Nups are poorly understood. NUCLEAR PORE ANCHOR (NUA) is a component of NPC. Loss of NUA leads to an increase in SUMO conjugates and pleiotropic developmental defects in Arabidopsis thaliana. Herein, we revealed that NUA is required for plant defense against multiple pathogens. NUCLEAR PORE ANCHOR associates with the transcriptional corepressor TOPLESS-RELATED1 (TPR1) and contributes to TPR1 deSUMOylation. Significantly, NUA-interacting protein EARLY IN SHORT DAYS 4 (ESD4), a SUMO protease, specifically deSUMOylates TPR1. It has been previously established that the SUMO E3 ligase SAP AND MIZ1 DOMAIN-CONTAINING LIGASE 1 (SIZ1)-mediated SUMOylation of TPR1 represses the immune-related function of TPR1. Consistent with this notion, the hyper-SUMOylated TPR1 in nua-3 leads to upregulated expression of TPR1 target genes and compromised TPR1-mediated disease resistance. Taken together, our work uncovers a mechanism by which NUA positively regulates plant defense responses by coordination with ESD4 to deSUMOylate TPR1. Our findings, together with previous studies, reveal a regulatory module in which SIZ1 and NUA/ESD4 control the homeostasis of TPR1 SUMOylation to maintain proper immune output.

Compensatory Mutations in GI and ZTL May Modulate Temperature Compensation in the Circadian Clock.

Circadian systems share the three properties of entrainment, free-running period, and temperature compensation (TC). TC ensures nearly the same period over a broad range of physiologically relevant temperatures; however, the mechanisms behind TC remain poorly understood. Here, we identify single point mutations in two key elements of the Arabidopsis circadian clock, GIGANTEA (GI) and ZEITLUPE (ZTL), which likely act as compensatory substitutions to establish a remarkably constant free-running period over a wide range of temperatures. Using near-isogenic lines generated from the introgression of the Cape Verde Islands (Cvi) alleles of GI and ZTL into the Landsberg erecta (Ler) background, we show how longer periods in the Cvi background at higher temperatures correlate with a difference in strength of the GI/ZTL interaction. Pairwise interaction testing of all GI/ZTL allelic combinations shows similar affinities for isogenic alleles at 22°C, but very poor interaction between GI (Cvi) and ZTL (Cvi) at higher temperature. In vivo, this would result in lower ZTL levels at high temperatures leading to longer periods in the Cvi background. Mismatched allelic combinations result in extremely strong or weak GI/ZTL interactions, indicating how the corresponding natural variants likely became fixed through epistatic selection. Additionally, molecular characterization of GI (Cvi) reveals a novel functional motif that can modulate the GI/ZTL interaction as well as nucleocytoplasmic partitioning. Taken together, these results identify a plausible temperature-dependent molecular mechanism, which contributes to the robustness of TC through natural variation in GI and ZTL alleles found on the Cape Verde Islands.

Kernel Architecture of the Genetic Circuitry of the Arabidopsis Circadian System.

A wide range of organisms features molecular machines, circadian clocks, which generate endogenous oscillations with ~24 h periodicity and thereby synchronize biological processes to diurnal environmental fluctuations. Recently, it has become clear that plants harbor more complex gene regulatory circuits within the core circadian clocks than other organisms, inspiring a fundamental question: are all these regulatory interactions between clock genes equally crucial for the establishment and maintenance of circadian rhythms? Our mechanistic simulation for Arabidopsis thaliana demonstrates that at least half of the total regulatory interactions must be present to express the circadian molecular profiles observed in wild-type plants. A set of those essential interactions is called herein a kernel of the circadian system. The kernel structure unbiasedly reveals four interlocked negative feedback loops contributing to circadian rhythms, and three feedback loops among them drive the autonomous oscillation itself. Strikingly, the kernel structure, as well as the whole clock circuitry, is overwhelmingly composed of inhibitory, rather than activating, interactions between genes. We found that this tendency underlies plant circadian molecular profiles which often exhibit sharply-shaped, cuspidate waveforms. Through the generation of these cuspidate profiles, inhibitory interactions may facilitate the global coordination of temporally-distant clock events that are markedly peaked at very specific times of day. Our systematic approach resulting in experimentally-testable predictions provides insights into a design principle of biological clockwork, with implications for synthetic biology.

Quantitative Circadian Phosphoproteomic Analysis of Arabidopsis Reveals Extensive Clock Control of Key Components in Physiological, Metabolic, and Signaling Pathways.

The circadian clock provides adaptive advantages to an organism, resulting in increased fitness and survival. The phosphorylation events that regulate circadian-dependent signaling and the processes which post-translationally respond to clock-gated signals are largely unknown. To better elucidate post-translational events tied to the circadian system we carried out a survey of circadian-regulated protein phosphorylation events in Arabidopsis seedlings. A large-scale mass spectrometry-based quantitative phosphoproteomics approach employing TiO2-based phosphopeptide enrichment techniques identified and quantified 1586 phosphopeptides on 1080 protein groups. A total of 102 phosphopeptides displayed significant changes in abundance, enabling the identification of specific patterns of response to circadian rhythms. Our approach was sensitive enough to quantitate oscillations in the phosphorylation of low abundance clock proteins (early flowering4; ELF4 and pseudoresponse regulator3; PRR3) as well as other transcription factors and kinases. During constant light, extensive cyclic changes in phosphorylation status occurred in critical regulators, implicating direct or indirect regulation by the circadian system. These included proteins influencing transcriptional regulation, translation, metabolism, stress and phytohormones-mediated responses. We validated our analysis using the elf4-211 allele, in which an S45L transition removes the phosphorylation herein identified. We show that removal of this phosphorylatable site diminishes interaction with early flowering3 (ELF3), a key partner in a tripartite evening complex required for circadian cycling. elf4-211 lengthens period, which increases with increasing temperature, relative to the wild type, resulting in a more stable temperature compensation of circadian period over a wider temperature range.

The F-box protein ZEITLUPE controls stability and nucleocytoplasmic partitioning of GIGANTEA.

Nucleocytoplasmic partitioning of core clock components is essential for the proper operation of the circadian system. Previous work has shown that the F-box protein ZEITLUPE (ZTL) and clock element GIGANTEA (GI) heterodimerize in the cytosol, thereby stabilizing ZTL. Here, we report that ZTL post-translationally and reciprocally regulates protein levels and nucleocytoplasmic distribution of GI in Arabidopsis. We use ectopic expression of the N-terminus of ZTL, which contains the novel, light-absorbing region of ZTL (the LOV domain), transient expression assays and ztl mutants to establish that the levels of ZTL, a cytosolic protein, help govern the abundance and distribution of GI in the cytosol and nucleus. Ectopic expression of the ZTL N-terminus lengthens period, delays flowering time and alters hypocotyl length. We demonstrate that these phenotypes can be explained by the competitive interference of the LOV domain with endogenous GI-ZTL interactions. A complex of the ZTL N-terminus polypeptide with endogenous GI (LOV-GI) blocks normal GI function, causing degradation of endogenous ZTL and inhibition of other GI-related phenotypes. Increased cytosolic retention of GI by the LOV-GI complex additionally inhibits nuclear roles of GI, thereby lengthening flowering time. Hence, we conclude that under endogenous conditions, GI stabilization and cytoplasmic retention occurs naturally through a LOV domain-mediated GI-ZTL interaction, and that ZTL indirectly regulates GI nuclear pools by sequestering GI to the cytosol. As the absence of either GI or ZTL compromises clock function and diminishes the protein abundance of the other, our results highlight how their reciprocal co-stabilization is essential for robust circadian oscillations.

Transcriptional corepressor TOPLESS complexes with pseudoresponse regulator proteins and histone deacetylases to regulate circadian transcription.

Circadian clocks are ubiquitous molecular time-keeping mechanisms that coordinate physiology and metabolism and provide an adaptive advantage to higher plants. The central oscillator of the plant clock is composed of interlocked feedback loops that involve multiple repressive factors acting throughout the circadian cycle. Pseudo response regulators (PRRs) comprise a five-member family that is essential to the function of the central oscillator. PRR5, PRR7, and PRR9 can bind the promoters of the core clock genes circadian clock associated 1 (CCA1) and late elongated hypocotyl (LHY) to restrict their expression to near dawn, but the mechanism has been unclear. Here we report that members of the plant Groucho/Tup1 corepressor family, topless/topless-related (TPL/TPR), interact with these three PRR proteins at the CCA1 and LHY promoters to repress transcription and alter circadian period. This activity is diminished in the presence of the inhibitor trichostatin A, indicating the requirement of histone deacetylase for full TPL activity. Additionally, a complex of PRR9, TPL, and histone deacetylase 6, can form in vivo, implicating this tripartite association as a central repressor of circadian gene expression. Our findings show that the TPL/TPR corepressor family are components of the central circadian oscillator mechanism and reinforces the role of this family as central to multiple signaling pathways in higher plants.

Arabidopsis CRY2 and ZTL mediate blue-light regulation of the transcription factor CIB1 by distinct mechanisms.

Plants possess multiple photoreceptors to mediate light regulation of growth and development, but it is not well understood how different photoreceptors coordinate their actions to jointly regulate developmental responses, such as flowering time. In Arabidopsis, the photoexcited cryptochrome 2 interacts with the transcription factor CRYPTOCHROME-INTERACTING basic helix-loop-helix 1 (CIB1) to activate transcription and floral initiation. We show that the CIB1 protein expression is regulated by blue light; CIB1 is highly expressed in plants exposed to blue light, but levels of the CIB1 protein decreases in the absence of blue light. We demonstrate that CIB1 is degraded by the 26S proteasome and that blue light suppresses CIB1 degradation. Surprisingly, although cryptochrome 2 physically interacts with CIB1 in response to blue light, it is not the photoreceptor mediating blue-light suppression of CIB1 degradation. Instead, two of the three light-oxygen-voltage (LOV)-domain photoreceptors, ZEITLUPE and LOV KELCH PROTEIN 2, but not FLAVIN-BINDING KELCH REPEAT 1, are required for the function and blue-light suppression of degradation of CIB1. These results support the hypothesis that the evolutionarily unrelated blue-light receptors, cryptochrome and LOV-domain F-box proteins, mediate blue-light regulation of the same transcription factor by distinct mechanisms.

PRR5 regulates phosphorylation, nuclear import and subnuclear localization of TOC1 in the Arabidopsis circadian clock.

Many core oscillator components of the circadian clock are nuclear localized but how the phase and rate of their entry contribute to clock function is unknown. TOC1/PRR1, a pseudoresponse regulator (PRR) protein, is a central element in one of the feedback loops of the Arabidopsis clock, but how it functions is unknown. Both TOC1 and a closely related protein, PRR5, are nuclear localized, expressed in the same phase, and shorten period when deficient, but their molecular relationship is unclear. Here, we find that both proteins interact in vitro and in vivo through their conserved N-termini. TOC1-PRR5 oligomerization enhances TOC1 nuclear accumulation two-fold, most likely through enhanced nuclear import. In addition, PRR5 recruits TOC1 to large subnuclear foci and promotes phosphorylation of the TOC1 N-terminus. Our results show that nuclear TOC1 is essential for normal clock function and reveal a mechanism to enhance phase-specific TOC1 nuclear accumulation. Interestingly, this process of regulated nuclear import is reminiscent of similar oligomeric pairings in animal clock systems (e.g. timeless/period and clock/cycle), suggesting evolutionary convergence of a conserved mechanism across kingdoms.

HSP90 functions in the circadian clock through stabilization of the client F-box protein ZEITLUPE.

The autoregulatory loops of the circadian clock consist of feedback regulation of transcription/translation circuits but also require finely coordinated cytoplasmic and nuclear proteostasis. Although protein degradation is important to establish steady-state levels, maturation into their active conformation also factors into protein homeostasis. HSP90 facilitates the maturation of a wide range of client proteins, and studies in metazoan clocks implicate HSP90 as an integrator of input or output. Here we show that the Arabidopsis circadian clock-associated F-box protein ZEITLUPE (ZTL) is a unique client for cytoplasmic HSP90. The HSP90-specific inhibitor geldanamycin and RNAi-mediated depletion of cytoplasmic HSP90 reduces levels of ZTL and lengthens circadian period, consistent with ztl loss-of-function alleles. Transient transfection of artificial microRNA targeting cytoplasmic HSP90 genes similarly lengthens period. Proteolytic targets of SCF(ZTL), TOC1 and PRR5, are stabilized in geldanamycin-treated seedlings, whereas the levels of closely related clock proteins, PRR3 and PRR7, are unchanged. An in vitro holdase assay, typically used to demonstrate chaperone activity, shows that ZTL can be effectively bound, and aggregation prevented, by HSP90. GIGANTEA, a unique stabilizer of ZTL, may act in the same pathway as HSP90, possibly linking these two proteins to a similar mechanism. Our findings establish maturation of ZTL by HSP90 as essential for proper function of the Arabidopsis circadian clock. Unlike metazoan systems, HSP90 functions here within the core oscillator. Additionally, F-box proteins as clients may place HSP90 in a unique and more central role in proteostasis.

ZEITLUPE is a circadian photoreceptor stabilized by GIGANTEA in blue light.

The circadian clock is essential for coordinating the proper phasing of many important cellular processes. Robust cycling of key clock elements is required to maintain strong circadian oscillations of these clock-controlled outputs. Rhythmic expression of the Arabidopsis thaliana F-box protein ZEITLUPE (ZTL) is necessary to sustain a normal circadian period by controlling the proteasome-dependent degradation of a central clock protein, TIMING OF CAB EXPRESSION 1 (TOC1). ZTL messenger RNA is constitutively expressed, but ZTL protein levels oscillate with a threefold change in amplitude through an unknown mechanism. Here we show that GIGANTEA (GI) is essential to establish and sustain oscillations of ZTL by a direct protein-protein interaction. GI, a large plant-specific protein with a previously undefined molecular role, stabilizes ZTL in vivo. Furthermore, the ZTL-GI interaction is strongly and specifically enhanced by blue light, through the amino-terminal flavin-binding LIGHT, OXYGEN OR VOLTAGE (LOV) domain of ZTL. Mutations within this domain greatly diminish ZTL-GI interactions, leading to strongly reduced ZTL levels. Notably, a C82A mutation in the LOV domain, implicated in the flavin-dependent photochemistry, eliminates blue-light-enhanced binding of GI to ZTL. These data establish ZTL as a blue-light photoreceptor, which facilitates its own stability through a blue-light-enhanced GI interaction. Because the regulation of GI transcription is clock-controlled, consequent GI protein cycling confers a post-translational rhythm on ZTL protein. This mechanism of establishing and sustaining robust oscillations of ZTL results in the high-amplitude TOC1 rhythms necessary for proper clock function.

Partners in time: EARLY BIRD associates with ZEITLUPE and regulates the speed of the Arabidopsis clock.

The circadian clock of the model plant Arabidopsis (Arabidopsis thaliana) is made up of a complex series of interacting feedback loops whereby proteins regulate their own expression across day and night. early bird (ebi) is a circadian mutation that causes the clock to speed up: ebi plants have short circadian periods, early phase of clock gene expression, and are early flowering. We show that EBI associates with ZEITLUPE (ZTL), known to act in the plant clock as a posttranslational mediator of protein degradation. However, EBI is not degraded by its interaction with ZTL. Instead, ZTL counteracts the effect of EBI during the day and increases it at night, modulating the expression of key circadian components. The partnership of EBI with ZTL reveals a novel mechanism involved in controlling the complex transcription-translation feedback loops of the clock. This work highlights the importance of cross talk between the ubiquitination pathway and transcriptional control for regulation of the plant clock.

Modelling of plant circadian clock for characterizing hypocotyl growth under different light quality conditions.

To meet the ever-increasing global food demand, the food production rate needs to be increased significantly in the near future. Speed breeding is considered as a promising agricultural technology solution to achieve the zero-hunger vision as specified in the United Nations Sustainable Development Goal 2. In speed breeding, the photoperiod of the artificial light has been manipulated to enhance crop productivity. In particular, regulating the photoperiod of different light qualities rather than solely white light can further improve speed breading. However, identifying the optimal light quality and the associated photoperiod simultaneously remains a challenging open problem due to complex interactions between multiple photoreceptors and proteins controlling plant growth. To tackle this, we develop a first comprehensive model describing the profound effect of multiple light qualities with different photoperiods on plant growth (i.e. hypocotyl growth). The model predicts that hypocotyls elongated more under red light compared to both red and blue light. Drawing similar findings from previous related studies, we propose that this might result from the competitive binding of red and blue light receptors, primarily Phytochrome B (phyB) and Cryptochrome 1 (cry1) for the core photomorphogenic regulator, CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1). This prediction is validated through an experimental study on Arabidopsis thaliana. Our work proposes a potential molecular mechanism underlying plant growth under different light qualities and ultimately suggests an optimal breeding protocol that takes into account light quality.

Frequency, composition and mobility of Escherichia coli-derived transposable elements in holdings of plasmid repositories.

By providing the scientific community with uniform and standardized resources of consistent quality, plasmid repositories play an important role in enabling scientific reproducibility. Plasmids containing insertion sequence elements (IS elements) represent a challenge from this perspective, as they can change the plasmid structure and function. In this study, we conducted a systematic analysis of a subset of plasmid stocks distributed by plasmid repositories (The Arabidopsis Biological Resource Center and Addgene) which carry unintended integrations of bacterial mobile genetic elements. The integration of insertion sequences was most often found in, but not limited to, pBR322-derived vectors, and did not affect the function of the specific plasmids. In certain cases, the entire stock was affected, but the majority of the stocks tested contained a mixture of the wild-type and the mutated plasmids, suggesting that the acquisition of IS elements likely occurred after the plasmids were acquired by the repositories. However, comparison of the sequencing results of the original samples revealed that some plasmids already carried insertion mutations at the time of donation. While an extensive BLAST analysis of 47 877 plasmids sequenced from the Addgene repository uncovered IS elements in only 1.12%, suggesting that IS contamination is not widespread, further tests showed that plasmid integration of IS elements can propagate in conventional Escherichia coli hosts over a few tens of generations. Use of IS-free E. coli hosts prevented the emergence of IS insertions as well as that of small indels, suggesting that the use of IS-free hosts by donors and repositories could help limit unexpected and unwanted IS integrations into plasmids.

Rapid assessment of gene function in the circadian clock using artificial microRNA in Arabidopsis mesophyll protoplasts.

Rapid assessment of the effect of reduced levels of gene products is often a bottleneck in determining how to proceed with an interesting gene candidate. Additionally, gene families with closely related members can confound determination of the role of even a single one of the group. We describe here an in vivo method to rapidly determine gene function using transient expression of artificial microRNAs (amiRNAs) in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. We use a luciferase-based reporter of circadian clock activity to optimize and validate this system. Protoplasts transiently cotransfected with promoter-luciferase and gene-specific amiRNA plasmids sustain free-running rhythms of bioluminescence for more than 6 d. Using both amiRNA plasmids available through the Arabidopsis Biological Resource Center, as well as custom design of constructs using the Weigel amiRNA design algorithm, we show that transient knockdown of known clock genes recapitulates the same circadian phenotypes reported in the literature for loss-of-function mutant plants. We additionally show that amiRNA designed to knock down expression of the casein kinase II ß-subunit gene family lengthens period, consistent with previous reports of a short period in casein kinase II ß-subunit overexpressors. Our results demonstrate that this system can facilitate a much more rapid analysis of gene function by obviating the need to initially establish stably transformed transgenics to assess the phenotype of gene knockdowns. This approach will be useful in a wide range of plant disciplines when an endogenous cell-based phenotype is observable or can be devised, as done here using a luciferase reporter.

Thinking outside the F-box: novel ligands for novel receptors.

The importance of regulated proteolysis in the physiology and development of plants is highlighted by the large number of genes dedicated to proteasome-dependent protein degradation. Within the SCF class of E3 ubiquitin ligases are more than 700 F-box proteins that act as recognition modules to specifically target their dedicated substrates for ubiquitylation. This review focuses on very recent studies indicating that some F-box proteins function as phytohormone or light receptors, which directly perceive signals and facilitate specific target-protein degradation to regulate downstream pathways. If this new connection between ligand-regulated proteolysis and signaling proves to be more extensive, an entirely new way of understanding the control of signal transduction is in the offing.

Post-Translational Mechanisms of Plant Circadian Regulation.

The molecular components of the circadian system possess the interesting feature of acting together to create a self-sustaining oscillator, while at the same time acting individually, and in complexes, to confer phase-specific circadian control over a wide range of physiological and developmental outputs. This means that many circadian oscillator proteins are simultaneously also part of the circadian output pathway. Most studies have focused on transcriptional control of circadian rhythms, but work in plants and metazoans has shown the importance of post-transcriptional and post-translational processes within the circadian system. Here we highlight recent work describing post-translational mechanisms that impact both the function of the oscillator and the clock-controlled outputs.

Cost-effective circadian mechanism: rhythmic degradation of circadian proteins spontaneously emerges without rhythmic post-translational regulation.

Circadian protein oscillations are maintained by the lifelong repetition of protein production and degradation in daily balance. It comes at the cost of ever-replayed, futile protein synthesis each day. This biosynthetic cost with a given oscillatory protein profile is relievable by a rhythmic, not constant, degradation rate that selectively peaks at the right time of day but remains low elsewhere, saving much of the gross protein loss and of the replenishing protein synthesis. Here, our mathematical modeling reveals that the rhythmic degradation rate of proteins with circadian production spontaneously emerges under steady and limited activity of proteolytic mediators and does not necessarily require rhythmic post-translational regulation of previous focus. Additional (yet steady) post-translational modifications in a proteolytic pathway can further facilitate the degradation's rhythmicity in favor of the biosynthetic cost saving. Our work is supported by animal and plant circadian data, offering a generic mechanism for potentially widespread, time-dependent protein turnover.