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Molecular mechanisms of light and clock-signaling pathways in Arabidopsis
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Development of plants extremely depends on reliable signaling mechanisms providing information on the actual state of the outer environment. The multilevel interaction of these mechanisms (e.g. light, clock and hormonal regulation) forms a regulatory network, which allows plants to respond adequately to the changes of the ambient environment.
Our research focuses on (i) the light signal transduction pathways mediated by the phytochrome photoreceptors, (ii) the structure and function of the plant circadian clock and (iii) the transcriptional regulation of genes involved in the biosynthesis of the steroidal phytohormones (brassinosteroids, BRs).
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Phytochrome-mediated signaling |
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Plants can sense the changes in the environmental light conditions by highly specialized photoreceptors. Among them, the red/far-red sensing phytochromes are the best characterized. In higher plants, phytochromes are encoded by a small multigene family; five genes (PHYA-PHYE) have been identified in Arabidopsis. The photosensory function of the molecule is based on its capacity for reversible interconversion between the red light-absorbing Pr form and the far-red light-absorbing Pfr form following sequential absorption of red and far-red light. Photosignal perception by the receptor is followed by conformational changes, and activation, through an as yet poorly understood mechanism, of signaling pathways leading to changes in the expression of genes that underlie developmental responses to light. We have demonstrated that light induces import of all phytochrome species into the nuclei, a process so far unique to plants. After they have been imported, they form nuclear speckles probably representing multi-protein complexes, which may directly or indirectly regulate the transcription of light-responsive genes. The light-regulated nucleocytoplasmic partitioning of phytochromes correlates well with the light quality dependence and kinetics of certain phytochrome-mediated light responses, therefore, this process may be a key element of light-induced signal transduction. Molecular characterization of the factors required for active retention of phytochromes in the cytosol in dark and for their light induced translocation into the nuclei is in progress. We also aim to identify components of phytochrome-containing nuclear speckles. |
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Figure 1. Phytochrome signaling pathways. Light absorption alters the conformation of phytochrome from Pr to the physiologically active Pfr form. The light induced conformation change is accompanied by autophosphorylation of the photoreceptor and phosphorylation of other proteins, like PKS1, which can induce intracellular signaling cascade (P: phosphorylated residues). Phytochrome also modifies the activity of other proteins, like NDPK2, which can result in the activation of another branch of phytochrome signaling. Light absorption is followed by the activation of heterotrimeric GTP-binding proteins (G), through an unknown mechanism, and leads to altered levels of cGMP and Ca2+. The modulation of these second messengers causes the activation of other factors (X and Y), putative components of transcriptional complexes that are required for light-dependent gene expression. The Pfr form of phytochrome is translocated to the nuclei and interacts with a set of transcriptional regulators constitutively present in the nuclei before light signal perception, such as PIF3. The primary targets of these transcription complexes are the promoters of various classes of transcription factor genes, like CCA1, LHY and HY5, causing a rapid induction (or repression) of the gene expression. It has been previously shown that PIF3 binds to the G-box element of CCA1 and LHY promoters and PHYB interacts with the promoter bound PIF3, inducing the expression of CCA1 and LHY. In the case of genes lacking G-box motive in their promoters, such as HY5, we propose that other, yet to be identified transcriptional regulators (indicated with PIFX) might be involved in this regulation. Dashed arrows indicate putative signaling pathways. |
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The plant circadian system |
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Many physiological and biochemical processes in plants exhibit endogenous rhythms with a period of about 24 hours. Endogenous oscillators, called circadian clocks regulate these rhythms. The circadian clocks are synchronized to the periodic environmental changes (e.g. day/night cycles) by specific stimuli; among these the most important is the light. Phytochromes are involved in setting the clock by transducing the light signal to the central oscillator, whereas the clock modulates phototransduction via the so-called gating phenomenon (Fig. 2).
Our studies on the light-controlled nucleo-cytoplasmic partitioning of various phytochrome species revealed that phototransduction, mediating entrainment of the circadian clock, is regulated by the circadian pacemaker itself. We have also demonstrated that the expression of all genes encoding phytochromes is also regulated by a circadian rhythm at the level of transcription. Taken together, these findings revealed new regulatory steps at the molecular level affecting light-induced signal transduction and question the basic features of generally accepted models regarding the role of phytochromes as photoreceptors in the function of the central circadian pacemaker. |
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Figure 2. Simplified model of the Arabidopsis circadian clock. The two MYB-related transcription factors, LHY and CCA1 form a reciprocal feedback loop with the pseudo response regulator, TOC1, which is postulated to be the basis of the oscillator mechanism, indicated with the grey circle. TOC1 positively regulates the expression of LHY and CCA1 by an unknown mechanism. An interaction between TOC1 and PIF3 molecules has been detected in yeast two-hybrid system, suggesting that TOC1 might regulate the expression of LHY and CCA1 via direct interaction with promoter bound PIF3, although the interaction of these two proteins has not yet been proved in planta. LHY and CCA1, in turn, negatively regulate the expression of TOC1 through binding to the promoter of TOC1 gene. This feedback loop results in the circadian expression of LHY, CCA1 and TOC1. Phytochrome is involved in the entrainment of the circadian clock upon light perception mediated by induction of LHY/CCA1 transcription through interaction with promoter-bound PIF3. The possible formation of a PHY/PIF3/TOC1 complex might represent an integrating point where light signals reach the circadian clock. The expression of phytochrome is also regulated by the clock, indicating the existence of a regulatory loop from the oscillator to the input pathways. CCA1 and LHY regulate the expression output genes, some of which are known to be induced by light through phytochrome action. The light induction of these genes is regulated by the oscillator, and ELF3, which interacts with PHYB (indicated with an arrow), has been shown to be involved in this "gating" process. ZTL, another component of the input pathways, can also interact with PHYB (indicated with an arrow) and influences the operation of the clock. Question marks indicate processes remaining to be elucidated. |
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In order to unravel new componets of the plant circadian clock, in collaboration with Andrew Millar's laboratory (University of Warwick, Coventry, UK) we have initiated a large-scale mutant screening program to isolate new circadian mutants in Arabidopsis. Characterization of these mutants is expected to help determine the number, specificity and hierarchy of circadian circuits existing in higher plants. |
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Transcriptional regulation of BR biosynthetic genes |
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In higher plants BRs control essential functions like elongation, photomorphogenesis, germination, flowering time and fertility. The steroid hormone environment is largely determined by the activity of BR biosynthetic genes, which are regulated primarily at the transcriptional level. In Arabidopsis, the transcript levels of all known BR biosynthetic genes are coordinately feedback-regulated by brassinolide, the end product of the synthesis pathway. In addition, each of these genes show characteristic developmental and organ-specific expression patterns. These regulatory mechanisms seem to be crucial for the homeostasis and morphogenic effects of the hormone.
The expression of the CPD (CONSTITUTIVE PHOTOPMORPHOGENESIS AND DWARFISM) gene, encoding a key cytochrome P450 enzyme of BR synthesis, also shows a marked fluctuation during the day. We study the activity of the CPD promoter in vivo, using transgenic Arabidopsis seedlings that contain this promoter fused to the firefly luciferase reporter gene. The changing expression levels are influenced by a combination of light, circadian, and hormonal regulation. We intend to carry out a genetic dissection of this complex control mechanism by using mutant backgrounds in which the light, circadian or BR control can be selectively abolished. Our aim is to determine the role of each of these regulatory factors in the daily cycling of CPD expression, and to find out if this oscillation results in periodic changes in the endogenous BR level. Characterization of the wavelength-dependence of the light effect will be instrumental in understanding whether the light control of BR biosynthesis is an important mediator of photomorphogenic effects such as etiolation or the shade-avoidance response. |
Szekeres, M., Németh, K., Koncz-Kálmán, Zs., Mathur, J., Kauschmann, A., Altmann, T., Rédei, G.P., Nagy, F., Schell, J. and Koncz, Cs. (1996). Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85: 171-182.
Kircher, S., Kozma-Bognár, L., Kim, L., Ádám, É., Harter, K., Schäfer, E. and Nagy, F. (1999). Light quality and quantity dependent nuclear translocation of phyA and phyB photoreceptors in higher plants. Plant Cell 11: 1445-1456.
Kozma-Bognár, L., Hall, A., Ádám, É., Thain, S., Nagy, F. and Millar, A. (1999). The circadian clock controls the expression pattern of the circadian input photoreceptor, phytochrome B. Proc. Natl. Acad. Sci. USA. 96: 14652-14657.
Nagy, F. and Schafer, E. (2000). Cytoplasmic and nuclear events for light regulated signalling in higher plants. EMBO J. 19: 157-163.
Nagy, F., Kircher, S. and Schafer, E. (2001). Intracellular trafficking of photoreceptors during light induced signal transduction in plants. J. of Cell Sci. 114: 475-480.
Toth, R., Kevei, E., Hall, A., Millar, A., Nagy, F. and Kozma-Bognar, L. (2001). Expression of phytochrome and cryptochrome genes is regulated by a circadian rhythm in Arabidopsis thaliana. Plant Physiology 127: 1607-1616.
Sweere, U., Eichenberg, K., Lohrmann, J., Mira-Rodado, V., Bauerle, I., Kudla, J., Nagy, F., Schafer, E. and Harter, K. (2001). Interaction of the response regulator ARR4 with the photoreceptor phytochrome B in modulating red light signaling. Science 294: 1108-1111.
Bancoş, S., Nomura, T., Sato, T., Molnár, G., Bishop, G.J., Koncz, C., Yokota, T., Nagy, F. and Szekeres, M. (2002). Regulation of transcript levels of the Arabidopsis cytochrome P450 genes involved in brassinosteroid biosynthesis. Plant Physiol. 130(1): 504-513.
Kircher, S., Gil, P., Kozma-Bognar, L., Fejes, E., Speth, V., Bauer, D., Adam, E., Schafer, E. and Nagy, F. (2002). Nucleo-cytoplasmic partitioning of the plant photoreceptors phytochrome A, B, C, D and E is differentially regulated by light and exhibits a diurnal rhythm. Plant Cell, 14(7): 1541-1555.
Doyle, M.R., Davis, S.J., Bastow, R.M., McWatters, H.G., Kozma-Bognár, L., Nagy, F., Millar, A.J. and Amasino, R.M. (2002). EARLY FLOWERING 4, a gene controlling circadian rhythms and flowering time in Arabidopsis thaliana. Nature, 419(6902): 74-77. 
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Dr. Ferenc NAGY