MOLECULAR MECHANISMS OF LIGHT AND CLOCK SIGNALING PATHWAYS IN ARABIDOPSIS

The development of plants depends on reliable signalling 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 changes in the ambient environment.
Our research focuses on (i) the signal transduction pathways induced by ultraviolet light, (ii) the molecular mechanism by which the red/far-red absorbing photoreceptors convert light into a biochemical signal, (iii) the structure and function of the plant circadian clock and (iv) the transcriptional regulation of genes involved in the biosynthesis of steroidal phytohormones (brassinosteroids, BRs).

Ultraviolet light induced signalling cascades

Ultraviolet light is a biologically important and dynamic component of sunlight. There is accumulating evidence that ultraviolet irradiation induces not only stress responses but, similarly to the visible part of the spectrum, it also functions as an environmental cue to regulate plant growth and development. We showed by microarray analysis that short, non-damaging pulses of UV induce a characteristic change in the expression profile of hundreds of genes and subsequently identified a number of key regulatory factors orchestrating this specific plant response. In collaboration with Roman Ulm’s laboratory in Freiburg, Germany we demonstrated that the bZIP transcription factor HY5, the multifunctional E3 ligase COP1 (constitutively photomorphogenic 1) and the UVR8 (ultraviolet resistant 8) protein play important roles in mediating UV-induced signalling. It has been demonstrated that UV light induces specific and rapid interaction of UVR8 and COP1 in planta and that this step is a very early and critical step in UV-induced signalling. We are interested in defining additional components of the UV-induced signalling network and in identifying specific cis-acting regulatory elements mediating UV-induced gene expression. UV and visible light induced signalling cascades share a number of key regulatory components, thus we intend to characterise how UV irradiation modifies phytochrome and circadian clock controlled signalling and ultimately plant growth and development.

Phytochrome-mediated signaling

Arabidopsis contains a small gene family encoding the phytochromes PHYA-PHYE. The photosensory function of the phytochrome molecule is based on its capacity to undergo reversible interconversion between the red light-absorbing Pr form and the far-red light-absorbing Pfr form. It has been shown that these conformational changes activate/deactivate signaling pathways, thereby leading to changes in the expression of about 2500-3000 genes that underlie developmental responses to light. We have demonstrated that light induces import of all phytochrome species into the nuclei in a wavelength and intensity dependent fashion and that phytochromes of nuclear localisation form nuclear bodies, which presumably represent multi-protein complexes (Figure 1). The biological function of these nuclear bodies is poorly understood. Our laboratory uses a number of experimental approaches to define the precise role of these phytochrome-containing nuclear bodies in light-induced signaling.

We have demonstrated that FHY1 (far-red elongated hypocotyl 1 and its close homolog FHL1 (fhy1-like) are necessary and sufficient to regulate light-induced nuclear import of PHYA in plants and in a cell-free system. Based on these observations and using FHY1 and PHYA as building blocks, we aim to create a synthetic light-regulated nuclear import/gene expression system, which can be used in plant, yeast or even mammalian cells.

Photoactivated PHYA is an extremely labile molecule with a half-life of ~20 min. The factors controlling this rapid degradation process are largely unknown. We have carried out a genetic screen and isolated mutants showing increased stability of an ectopically expressed PHYA-luciferase (PHYA-LUC) fusion protein in response to light (Figure 2). We have confirmed that light-induced degradation of the endogenous PHYA receptor is also inhibited in these mutants. Further characterization of the mutants and mapping of the mutations will reveal novel components/factors of light-controlled protein degradation in plants.

The circadian system in Arabidopsis thaliana

Circadian clocks are biological timing mechanisms; they generate a 24h basic oscillation and rhythmically regulate a wide range of processes in many organisms. In eukaryotes, the circadian oscillator relies on transcriptional/translational feedback loops operated by the clock genes and clock proteins. Circadian clocks are synchronized to the periodic environmental changes (e.g. day/night cycles) by specific stimuli; among these the most important is light. Phytochrome photoreceptors are involved in setting the clock by transducing the light signal to the central oscillator. We have showed that transcription of phytochrome genes and nuclear import of phytochrome receptor proteins are both regulated by the clock, indicating a two-way relationship between the components of the light input pathway and the oscillator.

In order to identify new components of the plant circadian clock, we have initiated a large-scale mutant screening program and isolated several new circadian mutants in Arabidopsis. Among them, the lip1-1 (light insensitive period 1) mutant displays novel circadian phenotypes arising from specific defects in the light input pathway to the oscillator. The LIP1 gene encodes a functional, plant-specific atypical small GTPase and therefore we postulate that it acts at the posttranscriptional level. Our current work is focused on elucidating the molecular mode of action of LIP1, which will allow us to integrate LIP1 in the model of the plant clock.

Characterization of additional mutants from the screen is in progress and is expected to help determine the number and hierarchy of circadian circuits existing in higher plants.

Developmental regulation of brassinosteroid biosynthesis

In higher plants brassinosteroids (BRs) control essential functions, such as 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 the biologically active end product of the synthesis pathway. In addition, at least two key genes of BR biosynthesis, CPD and the BR C-6 oxidase-encoding CYP85A2 are also under developmental, organ-specific, light-responsive and circadian control. Since BRs are not transported within the plant, these regulatory mechanisms are crucial for the homeostasis and morphogenic effects of the hormone.

We found that, in addition to the hormone gradient, light-dependent changes of susceptibility also influence BR responses. Our goal is to elucidate how, and to what extent BR accumulation and physiological sensitization contribute to the activation of BR signaling. To this end, we develop reporter lines allowing in vivo detection of BR distribution, and characterize the dependence of the hormone response on the expression level and functioning of BR signaling components.