Protein phosphorylation is a key regulatory mechanism in circadian systems. TIMING OF CAB EXPRESSION 1 (TOC1) is a core transcriptional repressor in the plant circadian system that is phosphorylated near its N terminus. Phenotype testing of TOC1 phosphosite mutants shows incomplete rescue of the short period toc1 mutant. We establish that TOC1 phosphorylation (particularly at S175) is necessary for optimal interaction with FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and PHYTOCHROME INTERACTING FACTOR 5 (PIF5) at the CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) promoter to down-regulate CCA1 expression. Expression of the closely related LATE ELONGATED HYPOCOTYL (LHY) also requires TOC1 but is independent of TOC1 phosphorylation, suggesting different TOC1-dependent gene repression mechanisms. We additionally show that TOC1 phosphorylation-dependent interactions at specific clock gene promoters selectively regulate these circadian system components more acutely than nonrhythmic genes. Our genome-wide analysis demonstrates that the TOC1 phosphostate is important for optimal chromatin presence and robust rhythmic gene expression.

Circadian clocks rely on transcriptional/translational feedback loops involving clock genes and their corresponding proteins. While the primary oscillations originate from gene expression, the precise control of clock protein stability plays a pivotal role in establishing the 24-hour circadian rhythms. Most clock proteins are degraded through the ubiquitin/26S proteasome pathway, yet the enzymes responsible for ubiquitination and deubiquitination remain poorly characterised. We identified a missense allele (ubp12-3, S327F) of the UBP12 gene/protein in Arabidopsis. Despite ubp12-3 exhibited a short period phenotype similar to that of a loss-of-function allele, molecular analysis indicated elevated protease activity in ubp12-3. We demonstrated that early flowering of ubp12 mutants is a result of the shortened circadian period rather than a direct alteration of UBP12 function. Analysis of protease activity of non-phosphorylatable (S327A, S327F) and phosphomimetic (S327D) derivatives in bacteria suggested that phosphorylation of serine 327 inhibits UBP12 enzymatic activity, which could explain the over-functioning of S327F in vivo. We showed that phosphomimetic mutations of the conserved serine in the Neurospora and human orthologues reduced ubiquitin cleavage activity suggesting that not only the primary structures of UBP12-like enzymes are phylogenetically conserved across a wide range of species, but also the molecular mechanisms governing their enzymatic activity.

Summary Arabidopsis PSEUDORESPONSE REGULATOR7 (PRR7) is a core component of the circadian oscillator which also plays a crucial role in freezing tolerance. PRR7 undergoes proteasome‐dependent degradation to discretely phase maximal expression in early evening. While its repressive activity on downstream genes is integral to cold regulation, the mechanism of the conditional regulation of the PRR7 abundance is unknown. We used mutant analysis, protein interaction and ubiquitylation assays to establish that the ubiquitin ligase adaptor, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), controls the protein accumulation pattern of PRR7 through direct protein–protein interactions at low temperatures. Freezing tolerance and electrolyte leakage assays show that PRR7 enhances cold temperature sensitivity, supported by ChIP‐qPCR at C‐REPEAT BINDING FACTOR1 (CBF1) and COLD‐REGULATED 15A (COR15A) promoters where PRR7 levels were higher in hos15 mutants. HOS15 mediates PRR7 turnover through enhanced ubiquitylation at low temperature in the dark. Under the same conditions, increased PRR7 association with the promoters of CBFs and COR15A in hos15 correlates with decreased CBF1 and COR15A transcription and enhanced freezing sensitivity. We propose a novel mechanism whereby HOS15‐mediated degradation of PRR7 provides an intersection between the circadian system and other cold acclimation pathways that lead to increased freezing tolerance.

This article is a Commentary on Zeng et al . (2023), 239 : 1253–1265 .

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.

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.

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.

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.

Achieving optimal plant growth is essential for the advancement of Arabidopsis thaliana (Arabidopsis) research. Over the last 20 years, the Arabidopsis Biological Resource Center (ABRC) has collected and developed a series of best-practice protocols, some of which are presented in this chapter. Arabidopsis can be grown in a variety of locations, growth media, and environmental conditions. Some mutant genotypes, natural accessions, and Arabidopsis relatives require strictly controlled growth conditions best provided by growth rooms, chambers, or incubators. Other lines can be grown in less-controlled greenhouse settings. Although the majority of lines can be grown in soil, certain experimental purposes require utilization of sterile solid or liquid growth media. These include the selection of primary transformants, identification of homozygous lethal individuals in a segregating population, or bulking of a large amount of plant material. The importance of controlling, observing, and recording growth conditions is emphasized and appropriate equipment for monitoring these conditions is listed. Proper conditions for seed harvest and preservation, as well as seed quality control procedures, are also described. In addition, plant transformation and genetic crosses, two of the methods that revolutionized Arabidopsis genetics, are discussed.

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.

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.

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.

Maturity date (MD), defined as the duration between the first calendar day of the year and maturity, and fruit development period (FDP), defined as the duration between full bloom and maturity, are highly variable in peach [Prunus persica (L.) Batsch]. There is a need to discover molecular markers associated with these traits in order to enhance the efficiency and reliability of breeding for extending the harvest season in peach. An association mapping population consisting of 132 peach accessions was phenotypically evaluated for MD and FDP, and genotypically characterized using the genotyping-by-sequencing (GBS) approach. The phenotypic and genotypic data collected were used to conduct a genome-wide association study (GWAS). The GWAS identified three SNPs on chromosome 4 that are significantly associated with both FDP and MD. These three SNPs covered a region of 43,067 bp; we referred to this region as the MD/FDP locus. Seven genes were identified in the MD/FDP locus. One or more of these genes is believed to regulate some aspect of maturity in peach. The data reported here is expected to aid in marker-assisted seedling selection (MASS) targeted towards widening peach germplasm for maturity, particularly early maturity.

Control of protein turnover is a key post-translational control point in the oscillatory network of the circadian clock. Some elements, such as TOC1 and PRR5 are engaged by a well-described F-box protein, ZEITLUPE, dedicated to their proteolytic turnover to shape their expression profile to a specific time of night. For most other clock components the regulation of their protein abundance is unknown, though turnover is often rapid and often lags the cycling of the respective mRNA. Here we report the design and results of an unbiased genetic screen in Arabidopsis to uncover proteolytic regulatory factors of PSEUDO-RESPONSE REGULATOR 7 (PRR7), a transcriptional repressor that peaks in the late afternoon. We performed EMS mutagenesis on a transgenic line expressing a PRR7::PRR7-luciferase (PRR7-LUC) translational fusion that accurately recapitulates the diurnal and circadian oscillations of the endogenous PRR7 protein. Using continuous luciferase imaging under constant light, we recovered mutants that alter the PRR7-LUC waveform and some that change period. We have identified novel alleles of ELF3 and ELF4, core components of the ELF3-ELF4-LUX Evening Complex (EC), that dampen the oscillation of PRR7-LUC. We report the characterization of two new hypomorphic alleles of ELF3 that help to understand the relationship between molecular potency and phenotype.

Circadian clocks play a pivotal role in orchestrating numerous physiological and developmental events. Waveform shapes of the oscillations of protein abundances can be informative about the underlying biochemical processes of circadian clocks. We derive a mathematical framework where waveforms do reveal hidden biochemical mechanisms of circadian timekeeping. We find that the cost of synthesizing proteins with particular waveforms can be substantially reduced by rhythmic protein half-lives over time, as supported by previous plant and mammalian data, as well as our own seedling experiment. We also find that previously enigmatic, cyclic expression of positive arm components within the mammalian and insect clocks allows both a broad range of peak time differences between protein waveforms and the symmetries of the waveforms about the peak times. Such various peak-time differences may facilitate tissue-specific or developmental stage-specific multicellular processes. Our waveform-guided approach can be extended to various biological oscillators, including cell-cycle and synthetic genetic oscillators.

The circadian clock is an endogenous timekeeping mechanism that can sustain oscillations in gene expression, metabolism and other aspects of physiology with a near 24 hour periodicity. Circadian systems require transcriptional and post-transcriptional processes to establish and maintain proper period and phase over a wide range of environmental conditions. This is especially true for poikilothermic organisms such as plants where the circadian system must face wide swings in ambient temperature yet sustain homeostasis of period and phase. The ultimate effectors of period control are proteins; transcription factors, other co-factors and enzymes. Most of the key clock proteins in plants such as the repressive myb-like transcription factors CCA1/LHY and the myb-related transcriptional activators of the REVIELLE (RVE) family oscillate robustly, indicating rapid protein turnover. The pseudoresponse regulator (PRR) family of proteins, unique to the plant clock, also oscillate strongly and undergo phase-dependent changes in phosphorylation. These and many of the other rhythmic clock proteins likely require additional modifications that govern their activity and stability, and additional factors are likely necessary for their maturation into fully functional polypeptides. Consistent with this notion, we have shown that the ubiquitous chaperone, HSP90, is necessary to maintain the normal period length in Arabidopsis. This differs from the effects of HSP90 depletion in metazoans, where period effects are absent or minimal and HSP90 appears to act as an integrator of processes leading to or from the central oscillator. In plants, HSP90 is specifically required for the maturation of ZEITLUPE (ZTL), an F-box protein required for the turnover of 2 essential clock proteins, TOC1 and PRR5. Reduced ZTL levels in HSP90-depleted plants affect only these 2 known SCF targets, and related PRR proteins are unaffected. Separately, we had previously shown that the unique plant protein, GIGANTEA (GI), is also necessary to sustain normal ZTL protein levels. gimutants cause constitutively low ZTL levels and GI overexpression results in ZTL overaccumulation. ZTL normally oscillates strongly in light/dark cycles and weakly in constant light, and it is the rhythmicity of GI, following on oscillations in GI transcript levels, that confers ZTL protein oscillations. Additionally, the ZTL-GI interaction is strongly enhanced by blue light, via the unique blue-light sensing LOV domain present at the N-terminus of ZTL. Our recent study now synthesizes these 2 previous reports into a single common mechanism, showing how HSP90 and GI work together to facilitate the maturation of ZTL. In vitro assays for chaperone holdase and foldase activities showed that GI possesses a general ability to both prevent protein denaturation and facilitate renaturation of a previously denatured enzyme. Importantly, when ZTLwas tested as a potential client protein, GI was similarly effective, showing holdase activity with wild-type ZTL but failing with mutant alleles of ZTL that also fail to interact with GI in vivo. Interestingly, in vitro foldase activity of GI on ZTL required the addition of HSP90/HSP70 to obtain full renaturation. These results were supported in planta using a ZTL-luciferase translational fusion (ZTL-LUC) to assess proper folding of the fusion protein in WT and gi mutant backgrounds. The maturation of ZTL-LUC was significantly reduced in the absence of GI or with ZTL point mutants that cannot interact with endogenous GI. Taken together, GI can now be seen as a novel co-chaperone of HSP90 that specifies ZTL as anHSP90 client. The ubiquity of HSP90 and its wide role in cellular proteostasis creates a challenge in understanding how specific clients are selected by HSP90 among the thousands of potential interactors. Co-chaperones help provide the specificity needed for that selection, but there is great variety in where they act during the HSP90 chaperone cycle. GI is very large (128 kDa) and interacts strongly with ZTL in vitro and very strongly with the N-terminal LOV domain in yeast. These findings, and the blue light enhanced interaction, suggest that GI does not require HSP90 for an initial ZTL interaction, and may instead help position the nascent ZTL polypeptide for entry into the HSP90 cycle. This may occur during early stages of folding, where HSP70 is likely engaged (Fig. 1). The large size of GI also suggests the possibility of additional proteins complexing with GI and ZTL, contributing to specifying the interaction. The in planta presence of a HSP90-GI-ZTL tripartite complex indicates that at least transiently all 3 elements are present together during the maturation process (Fig. 1). Loss of function gi mutants exhibit a wide range of phenotypes that extend beyond circadian dysfunction. FKF1, a member of the ZTL F-box protein family, also interacts with GI but

A LOV (Light, Oxygen, or Voltage) domain containing blue-light photoreceptor ZEITLUPE (ZTL) directs circadian timing by degrading clock proteins in plants. Functions hinge upon allosteric differences coupled to the ZTL photocycle; however, structural and kinetic information was unavailable. Herein, we tune the ZTL photocycle over two orders of magnitude. These variants reveal that ZTL complexes with targets independent of light, but dictates enhanced protein degradation in the dark. In vivo experiments definitively show photocycle kinetics dictate the rate of clock component degradation, thereby impacting circadian period. Structural studies demonstrate that photocycle dependent activation of ZTL depends on an unusual dark-state conformation of ZTL. Crystal structures of ZTL LOV domain confirm delineation of structural and kinetic mechanisms and identify an evolutionarily selected allosteric hinge differentiating modes of PAS/LOV signal transduction. The combined biochemical, genetic and structural studies provide new mechanisms indicating how PAS/LOV proteins integrate environmental variables in complex networks.

Most living organisms developed systems to efficiently time environmental changes. The plant-clock acts in coordination with external signals to generate output responses determining seasonal growth and flowering time. Here, we show that two Arabidopsis thaliana transcription factors, FAR1 RELATED SEQUENCE 7 (FRS7) and FRS12, act as negative regulators of these processes. These proteins accumulate particularly in short-day conditions and interact to form a complex. Loss-of-function of FRS7 and FRS12 results in early flowering plants with overly elongated hypocotyls mainly in short days. We demonstrate by molecular analysis that FRS7 and FRS12 affect these developmental processes in part by binding to the promoters and repressing the expression of GIGANTEA and PHYTOCHROME INTERACTING FACTOR 4 as well as several of their downstream signalling targets. Our data reveal a molecular machinery that controls the photoperiodic regulation of flowering and growth and offer insight into how plants adapt to seasonal changes.