light perception and plant development · •high irradiance response (hir) • the hir is used to...

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?