light perception and plant development · •high irradiance response (hir) • the hir is used to...
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Light perception and plant development
Electromagnetic irradiance : 350 bis 700 nm
- perception by eye (max: = 550 nm)
- cytosolic photoperception vs. perception via photosyntheticpigments
- photomrophogenesis vs. skotomorphogenesis
Photomorphogenesis
Light Dark
Skotomorphogenesis
Photomorphogenesis
Sinapis alba
SB21.17
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?
Monocotyledoneousseedlings
Alternative developmentalstrategy
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. Theyare all multimeric proteins containing a covalentlybound tetrapyrrole chromophore calledphytochromobilin. Phytochrome is involved in manyresponses 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 shadingresponse of certain small seeds.
• High irradiance response, which measures thebrightness of light, and is involved in the burialresponse 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 seedlingsunderneath soil or other plants) receive 10 timesmore far red light than red. Seeds respond byinhibiting germination until the canopy dies away.
• The mechanism of LFR relies on the fact thatphytochrome exists in two forms that areinterconverted by red and far-red light.
• Phytochrome is synthesised as inactive Pr whichabsorbs red light.
• Pfr is the active form, and absorbs far-red.
• The ratio of the two forms is a measure of the ratioof red to far red light.
• High [Pfr] indicates a lack of shading and thereforestimulates germination. High [Pr] indicates shadingand thus prevents germination.
• High irradiance response (HIR)
• The HIR is used to sense the presence of brightlight. It tells the plant when to perform such thingsas chlorophyll synthesis, plastid differentiation, de-etiolation of seedlings bursting through the soilsurface, and circadian rhythms (such as photonasticfolding of leaves or closing of flowers).
• The HIR requires several hours of bright light but isinsensitive to the red/far-red ratio.
• The LFR can't do this, because it saturates at lowlight intensities, and the Pr/Pfr ratio is determined bythe spectrum rather than the intensity of light.
HIR
LFR
Shaddow
> Pfr > Pr
long hypokotyl
field – plants at the edgesare shorter, becauseless shaded (containmore Pfr)
Phytochromes regulate gene expression
1979: Lhcb genes in barley
run-on assaystoday: 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 feedbackc.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- degradation of 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
Redlight
Far-redlight
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 cytoplasmand 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
Which are the chromophors?Do Phototropins and Cryptochromes have the same chromophors?
Flavine ?
Pterine ?
Carotene ?
Beyer
Phototropine
Phototropin-mediated reactions
Genotype
WT
phot1
phot2
phot1/phot2
Phototropismus
Chloroplast movement
Stomata opening
TIPS
Phototropin-mediated reactions
- Phototropismus- Chloroplasts movement- Stomata opening
One of the two phototropinsis responsible for low light responses,the other for high light responses.
WT
phot1
phot2
phot1/phot2
low light high light
Chloroplast movement Stomata openingPhototropismusGenotypeTIPS
1. phototropismBL 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
Ca2+
3. BL directs induces stomata opening
Guard cells
with
chlorophyll
fluorescence
…. Howeverstomata opening is regulated by many factors
LOV 2LOV 1 Ser/Thr-Kinase
Expression in E.coli
00.10.20.30.40.50.60.70.80.9
300 350 400 450 5000
0.10.20.30.40.50.60.70.80.9
300 350 400 450 500
FMNFMN
LOV 2LOV 1
Abs
orba
nce
Wavelength [nm]
PhototropinPhototropin –– structurestructure and and absorptionabsorption spectraspectra
FMN = Flavin
LOV = light/Oxygen/Voltage domain
Batschauer
N
N
CH3
CH3
O
O
R
NH
S
N
O HSH
NH
N
N
CH3
CH3
O
O
R
NH
H
S
NO
NHNH
Slow dark recovery
BL
Cystein
Cysteinyl-C(4a) adduct
5 4a 4a5
PhototropinPhototropin photoreactionphotoreaction
Flavin >
Redox reaction !!!!!!!
Batschauer
Cryptochrome
Cryptochrome-mediated responses
1. Inhibition of cell elongation2. Stimulation of anthocyanin biosynthesis3. Circadian rhythm
One of the two phototropinsis responsible for low light responses,the other for high light responses.
Domain structure
Pterin Flavin(FADH2)
ArabidopsisArabidopsis--CryptochromeCryptochrome
CRY11 500 681 AA
PHR1 500 AA
CRY21 500 612 AA
Pterin Flavin
Cryptochrome enthalten PterineFlavine als Chromophore.und
Batschaauer
Cryptochromes are present in many organismsand play a crucial role in circadian rhythm
Pterin Flavin
Marwan
Photolyases
Photolyases
Photo-reactivation by blue light
Photolyases are structurally similar to cryptochromes
Pterin/Deazaflavin FlavinMarwan
In summary…. In summary: BL and RL
Cryptochrome is centralfor 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 Q10 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- Schrittmacher
Temp.) (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 Absorptionsmaximum
Phase Funktion
Temperaturkompensation Flavonoidstoffwechsel
Entrainment Anthocyansynthese
Periode Funktion der Anthocyane
Schrittmacher/Oscillator Adaptation an UV-reiche
Input Regionen
Output
BL-Photorezeptor
feedback loopperiodtimelessclockcycleGonyaulax
Basics in plant signal transduction
Three types of receptors regulate signaling accross theplasma membrane
Signaling depends on calcium and cAMP/cGMP
Heterotrimeric G-proteins play a major role in animals, butare less important in plants
Heterotrimeric G-proteins activatethe adenylate cyclase in animals,
and a guanylate cyclase in plants
cAMP is replaced by cGMPas second messanger in
plants
Plant phospholipid signaling is quite different to animals, (phosphatidic acid, PLC and PLD, no IP3 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 identifysignaling 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
Land
sber
g
L/LC
olum
biaC/C
hete
rozy
gous
C/L
Cleaved amplified length polymorphism(CAPS) marker
Land
sber
g
L/L
Col
umbi
a
C/C
hete
rozy
gous
C/L
Land
sber
g
L/L
Col
umbi
a
C/C
hete
rozy
gous
C/L
restriction analysis
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 endsof the transposon form double stranded segments.
Volvox
= erste Leiche im Tierreich/
Pflanzenreich?