László Kozma-Bognár

Experimental validation of a predicted feedback loop in the multi‐oscillator clock of Arabidopsis thaliana

Our computational model of the circadian clock comprised the feedback loop between LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and TIMING OF CAB EXPRESSION 1 (TOC1), and a predicted, interlocking feedback loop involving TOC1 and a hypothetical component Y. Experiments based on model predictions suggested GIGANTEA (GI) as a candidate for Y. We now extend the model to include a recently demonstrated feedback loop between the TOC1 homologues PSEUDO‐RESPONSE REGULATOR 7 (PRR7), PRR9 and LHY and CCA1. This three‐loop network explains the rhythmic phenotype of toc1 mutant alleles. Model predictions fit closely to new data on the gi;lhy;cca1 mutant, which confirm that GI is a major contributor to Y function. Analysis of the three‐loop network suggests that the plant clock consists of morning and evening oscillators, coupled intracellularly, which may be analogous to coupled, morning and evening clock cells in Drosophila and the mouse.

The ELF4 gene controls circadian rhythms and flowering time in Arabidopsis thaliana

Many plants use day length as an environmental cue to ensure proper timing of the switch from vegetative to reproductive growth. Day-length sensing involves an interaction between the relative length of day and night, and endogenous rhythms that are controlled by the plant circadian clock. Thus, plants with defects in circadian regulation cannot properly regulate the timing of the floral transition. Here we describe the gene EARLY FLOWERING 4 (ELF4), which is involved in photoperiod perception and circadian regulation. ELF4 promotes clock accuracy and is required for sustained rhythms in the absence of daily light/dark cycles. elf4 mutants show attenuated expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a gene that is thought to function as a central oscillator component. In addition, elf4 plants transiently show output rhythms with highly variable period lengths before becoming arrhythmic. Mutations in elf4 result in early flowering in non-inductive photoperiods, which is probably caused by elevated amounts of CONSTANS (CO), a gene that promotes floral induction.

Light Quality–Dependent Nuclear Import of the Plant Photoreceptors Phytochrome A and B

The phytochrome (phy) family of plant photoreceptors controls various aspects of photomorphogenesis. Overexpression of rice phyA–green fluorescent protein (GFP) and tobacco phyB–GFP fusion proteins in tobacco results in functional photoreceptors. phyA–GFP and phyB–GFP are localized in the cytosol of dark-adapted plants. In our experiments, red light treatment led to nuclear translocation of phyA–GFP and phyB–GFP, albeit with different kinetics. Red light–induced nuclear import of phyB–GFP, but not that of phyA–GFP, was inhibited by far-red light. Far-red light alone only induced nuclear translocation of phyA–GFP. These observations indicate that nuclear import of phyA–GFP is controlled by a very low fluence response, whereas translocation of phyB–GFP is regulated by a low fluence response of phytochrome. Thus, light-regulated nucleocytoplasmic partitioning of phyA and phyB is a major step in phytochrome signaling.

Extension of a genetic network model by iterative experimentation and mathematical analysis.

Circadian clocks involve feedback loops that generate rhythmic expression of key genes. Molecular genetic studies in the higher plant Arabidopsis thaliana have revealed a complex clock network. The first part of the network to be identified, a transcriptional feedback loop comprising TIMING OF CAB EXPRESSION 1 (TOC1), LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), fails to account for significant experimental data. We develop an extended model that is based upon a wider range of data and accurately predicts additional experimental results. The model comprises interlocking feedback loops comparable to those identified experimentally in other circadian systems. We propose that each loop receives input signals from light, and that each loop includes a hypothetical component that had not been explicitly identified. Analysis of the model predicted the properties of these components, including an acute light induction at dawn that is rapidly repressed by LHY and CCA1. We found this unexpected regulation in RNA levels of the evening‐expressed gene GIGANTEA (GI), supporting our proposed network and making GI a strong candidate for this component.

Nucleocytoplasmic Partitioning of the Plant Photoreceptors Phytochrome A, B, C, D, and E Is Regulated Differentially by Light and Exhibits a Diurnal Rhythm

The phytochrome family of plant photoreceptors has a central role in the adaptation of plant development to changes in ambient light conditions. The individual phytochrome species regulate different or partly overlapping physiological responses. We generated transgenic Arabidopsis plants expressing phytochrome A to E:green fluorescent protein (GFP) fusion proteins to assess the biological role of intracellular compartmentation of these photoreceptors in light-regulated signaling. We show that all phytochrome:GFP fusion proteins were imported into the nuclei. Translocation of these photoreceptors into the nuclei was regulated differentially by light. Light-induced accumulation of phytochrome species in the nuclei resulted in the formation of speckles. The appearance of these nuclear structures exhibited distinctly different kinetics, wavelengths, and fluence dependence and was regulated by a diurnal rhythm. Furthermore, we demonstrate that the import of mutant phytochrome B:GFP and phytochrome A:GFP fusion proteins, shown to be defective in signaling in vivo, is regulated by light but is not accompanied by the formation of speckles. These results suggest that (1) the differential regulation of the translocation of phytochrome A to E into nuclei plays a role in the specification of functions, and (2) the appearance of speckles is a functional feature of phytochrome-regulated signaling.

Temporal repression of core circadian genes is mediated through EARLY FLOWERING 3 in Arabidopsis

Summary The circadian clock provides robust, ∼24 hr biological rhythms throughout the eukaryotes. The clock gene circuit in plants comprises interlocking transcriptional feedback loops, reviewed in [1], whereby the morning-expressed transcription factors CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) repress the expression of evening genes, notably TIMING OF CAB EXPRESSION 1 (TOC1). EARLY FLOWERING 3 (ELF3) has been implicated as a repressor of light signaling to the clock [2, 3] and, paradoxically, as an activator of the light-induced genes CCA1 and LHY [4, 5]. We use cca1-11 lhy-21 elf3-4 plants to separate the repressive function of ELF3 from its downstream targets CCA1 and LHY. We further demonstrate that ELF3 associates physically with the promoter of PSEUDO-RESPONSE REGULATOR 9 (PRR9), a repressor of CCA1 and LHY expression, in a time-dependent fashion. The repressive function of ELF3 is thus consistent with indirect activation of LHY and CCA1, in a double-negative connection via a direct ELF3 target, PRR9. This mechanism reconciles the functions of ELF3 in the clock network during the night and points to further effects of ELF3 during the day.

Forward Genetic Analysis of the Circadian Clock Separates the Multiple Functions of ZEITLUPE

The circadian system of Arabidopsis (Arabidopsis thaliana) includes feedback loops of gene regulation that generate 24-h oscillations. Components of these loops remain to be identified; none of the known components is completely understood, including ZEITLUPE (ZTL), a gene implicated in regulated protein degradation. ztl mutations affect both circadian and developmental responses to red light, possibly through ZTL interaction with PHYTOCHROME B (PHYB). We conducted a large-scale genetic screen that identified additional clock-affecting loci. Other mutants recovered include 11 new ztl alleles encompassing mutations in each of the ZTL protein domains. Each mutation lengthened the circadian period, even in dark-grown seedlings entrained to temperature cycles. A mutation of the LIGHT, OXYGEN, VOLTAGE (LOV)/Period-ARNT-Sim (PAS) domain was unique in retaining wild-type responses to red light both for the circadian period and for control of hypocotyl elongation. This uncoupling of ztl phenotypes indicates that interactions of ZTL protein with multiple factors must be disrupted to generate the full ztl mutant phenotype. Protein interaction assays showed that the ztl mutant phenotypes were not fully explained by impaired interactions with previously described partner proteins Arabidopsis S-phase kinase-related protein 1, TIMING OF CAB EXPRESSION 1, and PHYB. Interaction with PHYB was unaffected by mutation of any ZTL domain. Mutation of the kelch repeat domain affected protein binding at both the LOV/PAS and the F-box domains, indicating that interaction among ZTL domains leads to the strong phenotypes of kelch mutations. Forward genetics continues to provide insight regarding both known and newly discovered components of the circadian system, although current approaches have saturated mutations at some loci.

Functional interaction of the circadian clock and UV RESISTANCE LOCUS 8-controlled UV-B signaling pathways in Arabidopsis thaliana

Circadian clocks regulate many molecular and physiological processes in Arabidopsis (Arabidopsis thaliana), allowing the timing of these processes to occur at the most appropriate time of the day in a 24-h period. The accuracy of timing relies on the synchrony of the clock and the environmental day/night cycle. Visible light is the most potent signal for such synchronization, but light-induced responses are also rhythmically attenuated (gated) by the clock. Here, we report a similar mutual interaction of the circadian clock and non-damaging photomorphogenic UV-B light. We show that low-intensity UV-B radiation acts as entraining signal for the clock. UV RESISTANCE LOCUS 8 (UVR8) and CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) are required, but ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOG (HYH) are dispensable for this process. UV-B responsiveness of clock gene expression suggests that photomorphogenic UV-B entrains the plant clock through transcriptional activation. We also demonstrate that UV-B induction of gene expression under these conditions is gated by the clock in a HY5/HYH-independent manner. The arrhythmic early flowering 3-4 mutant showed non-gated, high-level gene induction by UV-B, yet displayed no increased tolerance to UV-B stress. Thus, the temporal restriction of UV-B responsiveness by the circadian clock can be considered as saving resources during acclimation without losing fitness.

Quantitative analysis of regulatory flexibility under changing environmental conditions

The circadian clock controls 24‐h rhythms in many biological processes, allowing appropriate timing of biological rhythms relative to dawn and dusk. Known clock circuits include multiple, interlocked feedback loops. Theory suggested that multiple loops contribute the flexibility for molecular rhythms to track multiple phases of the external cycle. Clear dawn‐ and dusk‐tracking rhythms illustrate the flexibility of timing in Ipomoea nil. Molecular clock components in Arabidopsis thaliana showed complex, photoperiod‐dependent regulation, which was analysed by comparison with three contrasting models. A simple, quantitative measure, Dusk Sensitivity, was introduced to compare the behaviour of clock models with varying loop complexity. Evening‐expressed clock genes showed photoperiod‐dependent dusk sensitivity, as predicted by the three‐loop model, whereas the one‐ and two‐loop models tracked dawn and dusk, respectively. Output genes for starch degradation achieved dusk‐tracking expression through light regulation, rather than a dusk‐tracking rhythm. Model analysis predicted which biochemical processes could be manipulated to extend dusk tracking. Our results reveal how an operating principle of biological regulators applies specifically to the plant circadian clock.

Diurnal Regulation of the Brassinosteroid-Biosynthetic CPD Gene in Arabidopsis

Plant steroid hormones, brassinosteroids (BRs), are essential for normal photomorphogenesis. However, the mechanism by which light controls physiological functions via BRs is not well understood. Using transgenic plants carrying promoter-luciferase reporter gene fusions, we show that in Arabidopsis (Arabidopsis thaliana) the BR-biosynthetic CPD and CYP85A2 genes are under diurnal regulation. The complex diurnal expression profile of CPD is determined by dual, light-dependent, and circadian control. The severely decreased expression level of CPD in phytochrome-deficient background and the red light-specific induction in wild-type plants suggest that light regulation of CPD is primarily mediated by phytochrome signaling. The diurnal rhythmicity of CPD expression is maintained in brassinosteroid insensitive 1 transgenic seedlings, indicating that its transcriptional control is independent of hormonal feedback regulation. Diurnal changes in the expression of CPD and CYP85A2 are accompanied by changes of the endogenous BR content during the day, leading to brassinolide accumulation at the middle of the light phase. We also show that CPD expression is repressed in extended darkness in a BR feedback-dependent manner. In the dark the level of the bioactive hormone did not increase; therefore, our data strongly suggest that light also influences the sensitivity of plants to BRs.