Növényi cirkadián óra-komponensek azonosítása és funkcionális jellemzése = Identification and functional characterization of components of the plant circadian clock

A cirkadián óra egy olyan genetikai alapokon nyugvó mechanizmus, amely képes kb. 24 órás periódushosszú oszcillációk létrehozására és ennek révén különböző életfolyamatok ritmikus szabályozására. Annak érdekében, hogy növényi óra újabb komponenseit fedezzük fel egy genetikai szűrés során cirkadián óra mutánsokat azonosítottunk Arabidopsisban. Genetikai térképezés majd az azt követő összehasonlító szekvenálás révén kimutattuk, hogy a mutánsok több, mint fele már ismert óragének új alléljeit reprezentálja. E mutánsok jellemzésével fontos új adatokhoz jutottunk több órafehérje funkcióját illetően. További 3 mutáció pozícióját mintegy 55-75 kbp kiterjedésű szakaszokra szűkítettük, amelyek szekvenálása folyamatban van. Mivel ezek a kromoszóma szakaszok nem tartalmaznak ismert óragént, a mutációk az óra új komponenseit érintik. A projekt legjelentősebb eredménye a lip1 (light insensitive period 1) mutáns azonosítása és jellemzése. Vad típusú növényekben az oszcillátor periódushossza függ a megvilágító fény intenzitásától: minél erősebb a fény, annál rövidebb a perióduszhossz. Kimutattuk, hogy a LIP1 fehérje alapvetően szükséges ehhez a szabályozáshoz, mivel hiányában a periódushossz minden fényviszony mellett azonos. Megállapítottuk, hogy a LIP1 gén egy funkcionális kis GTP-kötő fehérjét kódol, amely közvetett módon pozitívan szabályozza a GIGANTEA óragén transzkripcióját és feltehetően e kapcsolat révén hat az oszcillátor működésére. | Circadian clocks are genetic circuits capable of producing oscillations with ~ 24h period length and rhythmically regulating different biological processes. To learn more about the oscillatory mechanism of the plant circadian clock, we performed a genetic screen in Arabidopsis and identified several circadian mutants. Genetic mapping followed by comparative sequencing demonstrated that more than half of the mutants represent novel mutant alleles of known clock genes. Analysis of these new mutants provided valuable and novel data on the complex function of several clock proteins. Position of 3 additional mutations was delimited to 55-75 kbp regions, which are being sequenced. As these regions do not contain any known clock genes, the mutations must affect novel components of the clock. The most significant outcome of the project is the identification and characterisation of the lip1 (light insensitive period 1). In plants, there is an inverse relationship between the circadian period length and the intensity of the light: the period shortens with increasing light levels. LIP1 protein plays an essential role in this regulation, as the period length in lip1 is independent of light intensity. We isolated the LIP1 gene and demonstrated that it encodes a functional small GTP-binding protein. We showed that LIP1 positively regulates the transcription of the clock gene GIGANTEA that could be the mechanism by which LIP1 affects the function of the plant clock

A brasszinoszteroidok szerepének vizsgálata a növényi szervek morfogenezisének szabályozásában = The regulatory role of brassinosteroids in the morphogenic determination of plant organs

A brasszinoszteroidok (BR-ok) növényi szteroid hormonok, amelyek fontos szerepet játszanak az egyedfejlődési és reproduktív funkciók szabályozásában. Munkánk célja olyan tényezők azonosítása volt, amelyek a sejtek szenzitizálása, ill. a hormon felhalmozása révén befolyásolhatják a BR válaszreakciókat. Kimutattuk, hogy a feltételezett egyenletes kifejeződéssel szemben a BR receptort kódoló BRI1 gén differenciált szerv- és fejlődés-specifikus expressziót mutat. BRI1 promóter-riporter fúziókat hordozó transzgenikus Arabidopsis vonalainkban a fokozott hormonérzékenység szoros korrelációt mutatott a receptor erős kifejeződésével, jelezve a receptor sűrűség és a válaszreakció kialakulása közti kapcsolatot. Másrészt meghatároztuk az aktív BR formák korábban ismeretlen szintjét az Arabidopsis egyes szerveiben, igazolva, hogy a hormon felhalmozódás mértékét jórészt a bioszintetikus gének transzkripciós szintű szabályozása határozza meg. A sebesség-meghatározó CPD/CYP90A1 enzimről episztázis analízissel kimutattuk, hogy az a szintézis hatékonyságát közvetlenül a BR szintézis első reakcióját követően kontrollálja. Az anyagcsereút utolsó, még tisztázatlan enzim funkcióját karakterizálva meghatároztuk, hogy a CPD/CYP90A1 a szteroid váz C-3 pozíciójának oxidációjáért felelős. Ezzel egyúttal kísérletes bizonyítékot szolgáltattunk egy új, a korábban ismertnél hatékonyabb BR szintézisút in vivo jelentőségére. | Brassinosteroids (BRs) are steroidal plant hormones controlling morphogenesis and reproductive development. The aim of our project was to elucidate basic mechanisms that influence BR responses by modulating cellular susceptibility or accumulation of the biologically active hormone. We demonstrated that, in contrast to an earlier concept of ubiquitous expression, the BRI1 gene encoding the BR receptor shows differential organ-specific and developmental activity. Our BRI1 promoter-reporter constructs revealed that in Arabidopsis elevated BRI1 expression coincides with increased BR responsiveness, suggesting an important role for receptor abundance in the initiation of BR signaling. In a different approach we determined the hitherto unknown distribution of active BRs in the various organs of Arabidopsis, and provided evidence that the transcriptional control of BR biosynthetic genes efficiently regulates hormone accumulation. We have shown that CPD/CYP90A1 is a key enzyme of BR biosynthesis and, using epistasis analysis, we demonstrated that it controls intermediate flow immediately downstream of the first committed step of the BR pathway. Characterizing the last unknown enzyme function in the BR pathway, we clarified by in vitro enzyme assays that CPD/CYP90A catalyzes the C-3 oxidation of early BR intermediates. Based on this result we proposed an enzymologically well supported novel BR biosynthetic pathway

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

The Arabidopsis Zinc Finger Protein 3 interferes with ABA and light signaling in seed germination and plant development

Seed germination is controlled by environmental signals, including light and endogenous phytohormones. Abscisic acid (ABA) inhibits, whereas gibberellin promotes, germination and early seedling development, respectively. Here, we report that ZFP3, a nuclear C2H2 zinc finger protein, acts as a negative regulator of ABA suppression of seed germination in Arabidopsis (Arabidopsis thaliana). Accordingly, regulated overexpression of ZFP3 and the closely related ZFP1, ZFP4, ZFP6, and ZFP7 zinc finger factors confers ABA insensitivity to seed germination, while the zfp3 zfp4 double mutant displays enhanced ABA susceptibility. Reduced expression of several ABA-induced genes, such as RESPONSIVE TO ABSCISIC ACID18 and transcription factor ABSCISIC ACID-INSENSITIVE4 (ABI4), in ZFP3 overexpression seedlings suggests that ZFP3 negatively regulates ABA signaling. Analysis of ZFP3 overexpression plants revealed multiple phenotypic alterations, such as semidwarf growth habit, defects in fertility, and enhanced sensitivity of hypocotyl elongation to red but not to far-red or blue light. Analysis of genetic interactions with phytochrome and abi mutants indicates that ZFP3 enhances red light signaling by photoreceptors other than phytochrome A and additively increases ABA insensitivity conferred by the abi2, abi4, and abi5 mutations. These data support the conclusion that ZFP3 and the related ZFP subfamily of zinc finger factors regulate light and ABA responses during germination and early seedling development

Limited water stress modulates expression of circadian clock genes in Brachypodium distachyon roots

Organisms have evolved a circadian clock for the precise timing of their biological processes. Studies primarily on model dicots have shown the complexity of the inner timekeeper responsible for maintaining circadian oscillation in plants and have highlighted that circadian regulation is more than relevant to a wide range of biological processes, especially organ development and timing of flowering. Contribution of the circadian clock to overall plant fitness and yield has also long been known. Nevertheless, the organ- and species-specific functions of the circadian clock and its relation to stress adaptation have only recently been identified. Here we report transcriptional changes of core clock genes of the model monocot Brachypodium distachyon under three different light regimes (18:6 light:dark, 24:0 light and 0:24 dark) in response to mild drought stress in roots and green plant parts. Comparative monitoring of core clock gene expression in roots and green plant parts has shown that both phase and amplitude of expression in the roots of Brachypodium plants differ markedly from those in the green plant parts, even under well-watered conditions. Moreover, circadian clock genes responded to water depletion differently in root and shoot. These results suggest an organ-specific form and functions of the circadian clock in Brachypodium roots

Light and Temperature Signalling at the Level of CBF14 Gene Expression in Wheat and Barley

The wheat and barley CBF14 genes have been newly defined as key components of the light quality-dependent regulation of the freezing tolerance by the integration of phytochrome-mediated light and temperature signals. To further investigate the wavelength dependence of light-induced CBF14 expression in cereals, we carried out a detailed study using monochromatic light treatments at an inductive and a non-inductive temperature. Transcript levels of CBF14 gene in winter wheat Cheyenne, winter einkorn G3116 and winter barley Nure genotypes were monitored. We demonstrated that (1) CBF14 is most effectively induced by blue light and (2) provide evidence that this induction does not arise from light-controlled CRY gene expression. (3) We demonstrate that temperature shifts induce CBF14 transcription independent of the light conditions and that (4) the effect of temperature and light treatments are additive. Based on these data, it can be assumed that temperature and light signals are relayed to the level of CBF14 expression via separate signalling routes

Light Control of Salt-Induced Proline Accumulation is Mediated by Elongated Hypocotyl 5 in Arabidopsis

Plants have to adapt their metabolism to constantly changing environmental conditions, among which the availability of light and water is crucial in determining growth and development. Proline accumulation is one of the sensitive metabolic responses to extreme conditions; it is triggered by salinity or drought and is regulated by light. Here we show that red and blue but not far-red light is essential for salt-induced proline accumulation, upregulation of Delta 1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1) and downregulation of PROLINE DEHYDROGENASE 1 (PDH1) genes, which control proline biosynthetic and catabolic pathways, respectively. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the transcription factor ELONGATED HYPOCOTYL 5 (HY5) binds to G-box and C-box elements of P5CS1 and a C-box motif of PDH1. Salt-induced proline accumulation and P5CS1 expression were reduced in the hy5hyh double mutant, suggesting that HY5 promotes proline biosynthesis through connecting light and stress signals. Our results improve our understanding on interactions between stress and light signals, confirming HY5 as a key regulator in proline metabolism

Light Control of Salt-Induced Proline Accumulation Is Mediated by ELONGATED HYPOCOTYL 5 in Arabidopsis

Plants have to adapt their metabolism to constantly changing environmental conditions, among which the availability of light and water is crucial in determining growth and development. Proline accumulation is one of the sensitive metabolic responses to extreme conditions; it is triggered by salinity or drought and is regulated by light. Here we show that red and blue but not far-red light is essential for salt-induced proline accumulation, upregulation of Delta 1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1) and downregulation of PROLINE DEHYDROGENASE 1 (PDH1) genes, which control proline biosynthetic and catabolic pathways, respectively. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the transcription factor ELONGATED HYPOCOTYL 5 (HY5) binds to G-box and C-box elements of P5CS1 and a C-box motif of PDH1. Salt-induced proline accumulation and P5CS1 expression were reduced in the hy5hyh double mutant, suggesting that HY5 promotes proline biosynthesis through connecting light and stress signals. Our results improve our understanding on interactions between stress and light signals, confirming HY5 as a key regulator in proline metabolism

LIP1 Regulates the Plant Circadian Oscillator by Modulating the Function of the Clock Component GIGANTEA

Circadian clocks are biochemical timers regulating many physiological and molecular processes according to the day/night cycles. The function of the oscillator relies on negative transcriptional/translational feedback loops operated by the so-called clock genes and the encoded clock proteins. Previously, we identified the small GTPase LIGHT INSENSITIVE PERIOD 1 (LIP1) as a circadian-clock-associated protein that regulates light input to the clock in the model plant Arabidopsis thaliana. We showed that LIP1 is also required for suppressing red and blue light-mediated photomorphogenesis, pavement cell shape determination and tolerance to salt stress. Here, we demonstrate that LIP1 is present in a complex of clock proteins GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB 1 (TOC1). LIP1 participates in this complex via GUANINE EX-CHANGE FACTOR 7. Analysis of genetic interactions proved that LIP1 affects the oscillator via modulating the function of GI. We show that LIP1 and GI independently and additively regulate photomorphogenesis and salt stress responses, whereas controlling cell shape and photoperiodic flowering are not shared functions of LIP1 and GI. Collectively, our results suggest that LIP1 affects a specific function of GI, possibly by altering binding of GI to downstream signalling components