A növényi cirkadián óra beállításának molekuláris mechanizmusa = The molecular mechanism of entrainment of the plant circadian clock

A növényi cirkadián óra magját, más eukariotákhoz hasonlóan, az ún. óragének alkotják, amelyek egymás működését szabályozva egy önfenntartó, mintegy 24 órás ritmust alakítanak ki saját kifejeződésük szintjén. Az óra számos alapvető életfolyamat napszakos megjelenését szabályozza, így fontos, hogy az óra működése összhangban legyen a valós idővel. A beállítás során jön létre ez az összhang. A legfontosabb beállító külső jel a fény, amelynek intenzitása hűen követi a napszakok váltakozását. Munkánk során azt vizsgáltuk, hogy a fény milyen molekuláris mechanizmusok útján állítja be az óra fázisát. Kimutattuk, hogy a fénypulzusok által kiváltott elsődleges változás bizonyos óragének transzkripciójának tranziens indukciója, amelyet passzív módon követ az adott fehérjék mennyiségének emelkedése, ami végül az óra fázis-csúszását eredményezi. Mutánsok vizsgálatával megállapítottuk, hogy egyes óragének indukciója pozitív, ill. negatív módon szabályozza az óra fényre adott válaszának erősségét. Megállapításunkat egy új indukciós rendszer felépítésével és alkalmazásával is igazoltuk, melynek segítségével egyedei óragének tranziens indukciójának hatását vizsgálhattuk. Elsőként bizonyítottuk, hogy az UV-B fény a látható fényhez hasonló mechanizmus útján állítja be az órát és igazoltuk, hogy az óra fázisfüggő módon gátolja egyes UV-B válaszok megjelenését. Azonosítottuk a fitokróm B fotoreceptor azon doménjét, amely a fényjeleket közvetett módon az órához (óragénekhez) továbbítja. | Circadian clocks are biochemical timing mechanisms providing temporal regulation to a wide range of molecular and physiological processes so that these processes are scheduled to the most appropriate time of the day/night cycle. In plants the central oscillator relies on transcriptional/translational feedback loops operated by the clock genes/proteins. The central oscillator is synchronized to the day/night cycle mainly by light signals (input), whereas the rhythmic signal from the oscillator is relayed via the output pathway. In this project, we have investigated the molecular mechanisms, by which light signals reset the clock. We showed that the primary effect of light signals is the transient transcriptional activation of certain clock genes, followed by concomitant increase in protein levels leading to phase shifts of the clock. Analysis of clock mutants led to the identification of clock genes, whose induction represent positive or negative effects on the magnitude of the phase response of the clock. This was verified by the use of a novel gene expression system allowing separate induction of single (clock)genes. We have demonstrated first that UV-B light resets the clock through the same mechanism as visible light does, and provided evidences that the clock inhibits certain UV-B responses in a phase-dependent manner. Moreover, we identified the particular domain of the phytochrome B photoreceptor that relays resetting signals towards the clock

A switchable light-input, light-output system modelled and constructed in yeast

<p>Abstract</p> <p>Background</p> <p>Advances in synthetic biology will require spatio-temporal regulation of biological processes in heterologous host cells. We develop a light-switchable, two-hybrid interaction in yeast, based upon the Arabidopsis proteins PHYTOCHROME A and FAR-RED ELONGATED HYPOCOTYL 1-LIKE. Light input to this regulatory module allows dynamic control of a light-emitting LUCIFERASE reporter gene, which we detect by real-time imaging of yeast colonies on solid media.</p> <p>Results</p> <p>The reversible activation of the phytochrome by red light, and its inactivation by far-red light, is retained. We use this quantitative readout to construct a mathematical model that matches the system's behaviour and predicts the molecular targets for future manipulation.</p> <p>Conclusion</p> <p>Our model, methods and materials together constitute a novel system for a eukaryotic host with the potential to convert a dynamic pattern of light input into a predictable gene expression response. This system could be applied for the regulation of genetic networks - both known and synthetic.</p

Functional analysis of amino-terminal domains of the photoreceptor phytochrome B

At the core of the circadian network in Arabidopsis (Arabidopsis thaliana), clock genes/proteins form multiple transcriptional/translational negative feedback loops and generate a basic approximately 24-h oscillation, which provides daily regulation for a wide range of processes. This temporal organization enhances the fitness of plants only if it corresponds to the natural day/night cycles. Light, absorbed by photoreceptors, is the most effective signal in synchronizing the oscillator to environmental cycles. Phytochrome B (PHYB) is the major red/far-red light-absorbing phytochrome receptor in light-grown plants. Besides modulating the pace and phase of the circadian clock, PHYB controls photomorphogenesis and delays flowering. It has been demonstrated that the nuclear-localized amino-terminal domain of PHYB is capable of controlling photomorphogenesis and, partly, flowering. Here, we show (1) that PHYB derivatives containing 651 or 450 amino acid residues of the amino-terminal domains are functional in mediating red light signaling to the clock, (2) that circadian entrainment is a nuclear function of PHYB, and (3) that a 410-amino acid amino-terminal fragment does not possess any functions of PHYB due to impaired chromophore binding. However, we provide evidence that the carboxyl-terminal domain is required to mediate entrainment in white light, suggesting a role for this domain in integrating red and blue light signaling to the clock. Moreover, careful analysis of the circadian phenotype of phyB-9 indicates that PHYB provides light signaling for different regulatory loops of the circadian oscillator in a different manner, which results in an apparent decoupling of the loops in the absence of PHYB under specific light conditions

Host-secreted antimicrobial peptide enforces symbiotic selectivity in Medicago truncatula

Legumes engage in root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. In nodule cells, bacteria are enclosed in membrane-bound vesicles called symbiosomes and differentiate into bacteroids that are capable of converting atmospheric nitrogen into ammonia. Bacteroid differentiation and prolonged intracellular survival are essential for development of functional nodules. However, in the Medicago truncatula-Sinorhizobium meliloti symbiosis, incompatibility between symbiotic partners frequently occurs, leading to the formation of infected nodules defective in nitrogen fixation (Fix-). Here, we report the identification and cloning of the M. truncatula NFS2 gene that regulates this type of specificity pertaining to S. meliloti strain Rm41. We demonstrate that NFS2 encodes a nodule-specific cysteine-rich (NCR) peptide that acts to promote bacterial lysis after differentiation. The negative role of NFS2 in symbiosis is contingent on host genetic background and can be counteracted by other genes encoded by the host. This work extends the paradigm of NCR function to include the negative regulation of symbiotic persistence in host-strain interactions. Our data suggest that NCR peptides are host determinants of symbiotic specificity in M. truncatula and possibly in closely related legumes that form indeterminate nodules in which bacterial symbionts undergo terminal differentiation