Light perception and plant development
Electromagnetic irradiance : 350 bis 700 nm - perception by eye (max: = 550 nm) - cytosolic photoperception vs. perception via photosynthetic pigments - photomrophogenesis vs. skotomorphogenesis
SB21.17 Photomorphogenesis Skotomorphogenesis Photomorphogenesis Light Dark Sinapis alba
3 classes of cytoplasmic photoreceptors -phytochromes - absorption maxima: red/far-red -photoreversible - blue light/uv-a-photoreceptors - absorption maxima: blue/uv-a - heterogenous photoreceptors - phototropins and cryptochromes - UV-B photoreceptor - protective function - not characterized at the molecular level
PHYTOCHROMES
Dicotyledoneous seedlings - germination is normally light inducible - 3 criteria for photomorphogenesis - hypocotyl elongation - hock opening - cotyledon development - why such a developmental change? - isolation and characterization of mutants - involvement of photorecpetor?
Monocotyledoneous seedlings Alternative developmental strategy barley
RL illumination: Absorption by Pr Conversion from Pr to Pfr Pfr is physiologically active
photoreversible phytochromes
Domänenstruktur: N-Terminus: C- Terminus - Phy-Spezifität - Dimerisierungsdomäne - Chromophorbindung - Ubiquitinbindung - Signalweiterleitung
3 Arten der Phytochromantworten - Niederflußantwort (low fluence response) RL durch DR revertierbar Absorpitonsmaximum bei Dauerbestrahlung im RL Phytochrom B (und aufwärts) - Hochintensitätsrekation (Phytochrom A) Absorptionsmaximum im DR Wirkung bei Dauerbestrahlung energieflußabhängig - Niedrigstflußreaktion (very low fluence response) benötigt < 0.1% Pfr nicht revertierbar
Phytochromes summary Phytochromes are blue-green pigments found in all green plants. They sense red and far-red light. They are all multimeric proteins containing a covalently bound tetrapyrrole chromophore called phytochromobilin. Phytochrome is involved in many responses to light. The two most well studied are the: Low fluence response, which measures the ratio of red to far-red light, and is involved in the shading response of certain small seeds. High irradiance response, which measures the brightness of light, and is involved in the burial response of germinated seeds (etiolation).
Low fluence response (LFR) Lettuce seed germination requires light. Red light stimulated germination but far red reversed the effect of red light and inhibited germination. Thus phytochrome appeared to be measuring the red to far-red ratio, and required only about 1 µmol m 2 of photons. This is useful because shaded plants (and seedlings underneath soil or other plants) receive 10 times more far red light than red. Seeds respond by inhibiting germination until the canopy dies away.
The mechanism of LFR relies on the fact that phytochrome exists in two forms that are interconverted by red and far-red light. Phytochrome is synthesised as inactive P r which absorbs red light. P fr is the active form, and absorbs far-red.
The ratio of the two forms is a measure of the ratio of red to far red light. High [P fr ] indicates a lack of shading and therefore stimulates germination. High [P r ] indicates shading and thus prevents germination.
High irradiance response (HIR) The HIR is used to sense the presence of bright light. It tells the plant when to perform such things as chlorophyll synthesis, plastid differentiation, deetiolation of seedlings bursting through the soil surface, and circadian rhythms (such as photonastic folding of leaves or closing of flowers). The HIR requires several hours of bright light but is insensitive to the red/far-red ratio. The LFR can't do this, because it saturates at low light intensities, and the P r /P fr ratio is determined by the spectrum rather than the intensity of light.
HIR LFR
Shaddow > Pfr > Pr long hypokotyl field plants at the edges are shorter, because less shaded (contain more Pfr)
Phytochromes regulate gene expression 1979: Lhcb genes in barley run-on assays today: more than 1500 genes are phytochrome-regulatded Photosynthesis genes N/S metabolism Genes for plastid proteins pigment genes (Anthocyanin and Flavonoids) Phytochrome contols the expression of its own gene (phya) negative feedback c.f. Protochlorophyllide-Oxidoreduktase
Light labile Phytochrome A vs. Light stabile Phytochromes B-E - Synthesis of Phytochrome A in etiolated seedlings - transition from dark to light - downregulation of transcription of phya -degradationof phya message - degradation of phya protein - developmenal control is shifted from PhyA to PhyB-E
Mutants - Chromophor mutants (all phytochromes are affected) - Apoprotein mutants - loss of function mutants (e.g. defect in signaling component) - gain of function mutants (e.g. constitutive active signaling component) Phenotypes dark phenotype in light light phenotype in dark
Photoreceptor mutants of Arabidopsis thaliana Red light Far-red light
Alternative: Phytochrome migrates into the nucleus Interaction with partner proteins Pfr-form migrates, Pr-form stays in cytoplasm Active retardation of Pr-form General principles of distribution of proteins between cytoplasm and nucleus - transcription factors - steroid hormone receptors -cryptochromes
blue/uv-a light photoreceptors - phototropin 1 und 2 - cryptochrome 1 und 2 -(photolyases)
blue light - 400 500 nm - phototropismus - Overlapping functions with phytochromes - hypocotyl elongation - gene expression - pigment synthesis - stomata movementung - phototaxis
Different action spectra Phototropismus Avena Chloroplast movement DNA-photoreactivation
Beyer Which are the chromophors? Do Phototropins and Cryptochromes have the same chromophors? Flavine? Pterine? Carotene?
Phototropine
TIPS Phototropin-mediated reactions WT phot1 Phototropismus Chloroplast movement phot2 Stomata opening phot1/ phot2 Genotype
Phototropin-mediated reactions - Phototropismus - Chloroplasts movement - Stomata opening One of the two phototropins is responsible for low light responses, the other for high light responses.
TIPS low light high light WT phot1 phot2 phot1/ phot2 Genotype Phototropismus Chloroplast movement Stomata opening
1. phototropism BL directs auxin to the shaded side to induce phototropic curvature
Phototropism of the sporangiophore (fruiting body) of the zygomycete, Phycomyces blakesleeanus
2. BL induces chloroplast movement
Mougeotia Chloroplast movement light position dark position In low light phytochrome-controlled Polarized light Plasma membrane Mikrotobuli Ca 2+
3. BL directs induces stomata opening
Guard cells with chlorophyll fluorescence
. However stomata opening is regulated by many factors
Batschauer Phototropin structure and absorption spectra LOV 1 LOV 2 Ser/Thr-Kinase FMN LOV 1 FMN LOV 2 Expression in E.coli Absorbance 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 300 350 400 450 500 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 300 350 400 450 500 Wavelength [nm] FMN = Flavin LOV = light/oxygen/ Voltage domain
Batschauer Phototropin photoreaction Flavin > CH 3 CH 3 O N Cystein R N O BL R CH 3 N O 5 4a NH N H 5 4a N CH 3 N H SH S H O O S Slow dark recovery O N NHH Cysteinyl-C(4a) adduct Redox reaction!!!!!!!
Cryptochrome
Cryptochrome-mediated responses 1. Inhibition of cell elongation 2. Stimulation of anthocyanin biosynthesis 3. Circadian rhythm One of the two phototropins is responsible for low light responses, the other for high light responses.
Domain structure Pterin Flavin (FADH 2 )
Batschaauer Arabidopsis-Cryptochrome Pterin CRY1 Flavin 1 500 681 AA CRY2 1 500 612 AA PHR Cryptochrome enthalten Pterine 1 500 AA und Flavine als Chromophore.
Marwan Cryptochromes are present in many organisms and play a crucial role in circadian rhythm Pterin Flavin
Photolyases
Photolyases Photo-reactivation by blue light
Marwan Photolyases are structurally similar to cryptochromes Pterin/Deazaflavin Flavin
. In summary: BL and RL
Cryptochrome is central for the control of circadian rhythm
Periodenlänge (Abstand von peak 1 zu peak 2): Mutanten bei Drosophila, Neurospora, Chlamydomonas, Arabidopsis, Maus per Gen in Drosophila, perl, pers, pera (lang, kurz, arhythmisch) Austausch von bestimmte AA beim per Gen Temperaturkompensation Q 10 von 0.8 bis 1.3 Phase(responsekurve) PRC: Verschiebung der peaks Entrainment: Einstellung auf neuen Licht/Dunkelrhythmus Circadian/diurnal
Input Oscillator Output (Licht, endogener response -Phy, Cry- Temp.) Schrittmacher (z. B..per, tim, frq, toc)
Fliegen- und Säugerbeispiel 2 Proteine in Cytosol: period & timeless (cryptochrome) Interaktion über PAS-Domäne tagesabhängige Phosphorylierung Kerntranslokation De-aktivierung der TF clock und cycle (bhlh mit PAS) clock und cycle: binden an E box der per/tim Per/cry Gene. Mechanismus: feedback loop Cryptochrome: Arabidopsis/Drosophila: BL-Photorezeptor, beteiligt am circadianen Eingang Säuger: Oscillatorkomponente
Gonyaulax polyedra: - nicht transkriptional -translational Biolumineszenz in der Nacht Zellteilung bei Morgendämmerung Photosynthese am Tag Zellaggregration am Tag - Beispiel: Luciferinbindeprotein, Luciferase, Peridin-/Chlorophyll-Biindeprotein
UV-B Photorezeptor - molekular nicht charakterisiert - Absorptionsmaximum bei 290 nm (UV-B) - Schutzfunktion - Induktion von Flavonen/Flavonoiden/Anthocyane - Modellgene: Schlüssenenzyme dieses Syntheseweges: (PAL, CHS, etc.) - Ablagerung in der Vakuole - Klassische Untersuchungsbeispiele: - Hirsevarietät: Strickte UV-B Abhängigkeit der Anthocyansynthese - Petersillie-Zellkulturen - Petunien (Blütenfarbstoffe, Freisetzungsversuche)
Adaptation an UV-B reiche Regionen (Berghöhen, dünne Ozonschicht) - Hemmung des Hypokotylwachstums - Stimulation der Anthocyansynthese
Circadiane Rhythmik UV-B-Photorezeptor Circadian Phase Temperaturkompensation Entrainment Periode Schrittmacher/Oscillator Input Output BL-Photorezeptor feedback loop period timeless clock cycle Gonyaulax Absorptionsmaximum Funktion Flavonoidstoffwechsel Anthocyansynthese Funktion der Anthocyane Adaptation an UV-reiche Regionen
Basics in plant signal transduction
Three types of receptors regulate signaling accross the plasma membrane
Signaling depends on calcium and camp/cgmp
Heterotrimeric G-proteins play a major role in animals, but are less important in plants
Heterotrimeric G-proteins activate the adenylate cyclase in animals, and a guanylate cyclase in plants
camp is replaced by cgmp as second messanger in plants
Plant phospholipid signaling is quite different to animals, (phosphatidic acid, PLC and PLD, no IP 3 receptor at ER)
The two component system plays an important role in plant hormone signaling histidine kinase & response regulator
Ca signaling is very complex in plants - Source of Ca (external, internal stores) - CDPK - more than 100 Ca-binding pregulatory proteins (network) - CaCaMK are located in cytoplasm and nucleus - Ca signatures differ
Ca signature determines response patterns
MAPKs play important roles in plant defense
Genetic approaches to identify signaling processes in plants Isolation of mutants Identification of mutated genes Characterization of gene product
Isolation, Herstellung und Untersuchung von Mutanten I - natürliche Mutanten - chemische Mutagenese (Ethylmethansulfonat) - Ethylierung von G -G > A - Mutationen durch Röntgenstrahlen - Insertionsmutagenese - statistische Insertionen von Fremd-DNA -T-DNA tags - transponierbare Elemente -Samen-oder Pollenmutagenese
Isolation, Herstellung und Untersuchung von Mutanten II Insertionsmutagenese - bekannte Insertion - flankierende Regionen werden mittels PCR amplifiziert - Sequenzen mit Datenbanken abstimmen - Insertionsort ermitteln mittels Datenbanken - international verfügbare Insertionslinien - Vor-/Nachteil dieses Verfahrens: das gesamte Genprodukt fehlt
Isolation, Herstellung und Untersuchung von Mutanten III chemische/physikalische Mutagenese - Identifikation der Punktmutation/Deletion/Rearrangierung über markergestützte Kartierungen - Kreuzung mit Ökotypen - Kartierungen über RFLPs (Polymorphismen) - Verwendung von international verfügbaren Markers oder eigener Marker - Vor-/Nachteil dieses Verfahrens: - Mutation liegt in einem Epitop - Isolation mehrerer Mutanten
Beispiel für eine EMS Mutante im Photosystem II
Kreuzung mit einem anderen Ökotyp (Mutation im Ökotyp I, Kreuzung mit Ökotyp II) - Austausch der Chromosomen - Identifikation von Nachkommen, bei denen alle Chromsomomen ohne Mutation vom Ökotyp II sind - Austausch der genetischen Information auf dem verbliebenen Chromsosm vom Ökotyp I durch cross-over - soviel Ökotyp I-DNA wie möglich durch Ökotyp II-DNA ersetzen - zurück bleibt: kurzes Ökotyp I-DNA Segment mit Mutation Analyse der Nachkommen: - Unterscheidung von Ökotyp I und II DNA durch Marker - Identifikation der Mutation durch Sichten der Nachkommen
Marker: alles, was eine Zuordnung eines Merkmals zu einem Ökotyp erlaubt morphologische Marker: Blütenfarbe, Stengellänge, Ertrag Molekulare Marker: alles, was die Zuordnung eines DNA- Abschnitts zu einem Ökotyp erlaubt z. B. Restriktionslängenpolymorphismus (RFLP) Restriktionsenzym schneidet in der DNA eines Ökotyps, aber nicht in der DNA des anderen.
Simple sequence length polymorphism (SSLP) marker Landsberg Columbia heterozygous L/L C/C C/L
Cleaved amplified length polymorphism (CAPS) marker Landsberg Columbia heterozygous Landsberg Columbia heterozygous L/L C/C C/L restriction analysis L/L C/C C/L
Colinearity of genomes
Transposons/Retrotransposons
Transposable element in maize
A transposon is a piece of DNA that is flanked by two insertion elements (IS elements) oriented opposite (a palindrome). At the top: within a DNA double strand. At the bottom: when denaturing the double strand (just one of them is shown). The inversely oriented IS elements (insertion elements) at the ends of the transposon form double stranded segments.
Volvox = erste Leiche im Tierreich/ Pflanzenreich?