植物开花时间控制的分子机理

Molecular mechanism of plant flowering time control

第十章 基因和发育第二讲

By Hongwei Guo, Peking University, 2008.12.18

Transition to reproduction

Vegetative phase

Reproductive phase

When to flower is a matter of the survival

of species

Two major pathways regulating floral transition

Photoperiodic flowering (光周期开花 ): day-length dependent flowering time control

Vernalization (春化 ): cold-promoted flowering

(“Maryland Mammoth” cultivar of tobacco)

Floral induction of leaves of Short Day plant Perrila crispa

“Something” must be produced in leaves and “move” to the meristem

Long journey to identify “Florigen” began

(Lang and Zeevart)

Multiple grafting experiment with Perilla

Long distance transport---- thru vascular tissue

• the flowering signal is generated in the leaf

• the signal goes one way: from the leaf to the apex

• Grafting transmittable

The flowering signal: florigen vegetative or reproductive growth?

SAM

Florigen

Florigen

Florigen

Photoperiodic flowering

Two essential questions:

1. How does the leaf measure day-length?

2. What is the florigen?

Genetics provides the answers

Arabidopsis: Long Day PlantFlowering is induced by Long Day (LD)

Certain late flowering mutants are blind to photoperiod

cryptochrome 2 (cry2)phytochrome A (phyA)contstans (co) flowering locus t (ft)

Cry2: blue light receptorPhyA: Far-red light receptorCO: transcriptional co-activatorFT: transcriptional co-activator (?)

3 wavelengths (Blue, Red, Far-red) are important in regulating plant growth and development

Phytochromes (光敏色素）： Red/Far-red light receptorsCryptochromes (隐花色素）： Blue light receptors

phyA also absorbs blue light

SignalTrans-duction

Light has a dual role in this model: - entrains the circadian oscillation of light- and dark-sensitive phases- directly required for the production of the signal.

(originally proposed by Boenning, 1936)

PRR: photoperiod response regulator

PRR: photoperiod response regulator

CO is essential for photoperiodic flowering, as co mutant is late flowering and almost a day-neutral plant. It encodes a transcriptional regulator.

LFY

A, B, C, E class genes

（春化）Is co the PRR that measures daylength?

CO is required for FT expression

FT mRNA levels determine flowering time

SD: late floweringLD: early floweringphyBDE: early flowering cry2: late flowering

When CO mRNA peaks at midnight in SDs, COP1 is predominantly localized in the nucleus. CRY interacts with COP1 but is not able to repress its activity. COP1–CO interaction results in ubiquitination and degradation of CO (A), whereas when CO mRNA peaks in the afternoon in LDs, light activation of CRY during the daytime might mediate translocation of COP1 from nucleus to cytoplasm. Consequently, CO is able to accumulate and activate the transcription of FT to promote flowering (B). U, ubiquitin.

Cry inhibits CO protein degradation in light

Molecular mechanism of photoperiodic flowering

Photoperiod-dependent activation of CO protein and FT mRNA

CO mRNA is regulated by circadian clock.CO protein is stabilized by light

CDF1: circadian dependent factor

Therefore, FT expression is activated only in long day

CO protein measures day-length

FT mRNA level determines flowering time

FT (or Hd3a) is a floral activator both in LD and SD plants

Long Day Plant Short Day Plant

CO in Long Day plants and similar proteins (Hd1) in Short Day plants are regulated in opposite ways

(Kobayashi & Weigel, 2007)

retr

2005

Heating the leaf of pHSP::FT transgenic plants can promote flowering, and FT mRNA can be detected in SAM

However, the mRNA hypothesis was challenged in 2006 by a PNAS paper, the original Science paper was retracted in 2007.

5 more papers have been published in 2007 – all argued that FT protein is the florigen, in at least 5 different plant species: Arabidopsis, rice, tomato, tobacco, pumpkin.

Yuval Eshed‘s lab in Israel cloned tomato FT gene from a tomato mutant, sft. They overexpressed SFT in tomato, resulting in early flowering, but they found little transgenic SFT mRNA in the apical meristem 。

SAM leaves

Where in the vascular tissue is FT mRNA orFT protein locate (and migrate)?

Phloem sap: solutes migrating thru phloem (sieve tube)

FT protein is a “florigen”

CO – transcription factor in leaves, respond to day-length (photoperiod sensor)

FT – RAF kinase inhibitor protein, travels in phloem from leaf to SAM, target of CO

So……..

Photoperiodic flowering in plants

FT protein

FT protein

Hd3a protein

GI

Besides the photoperiod-dependent regulation, floral transition is under controls of many other cues.

67

Vernalization (春化 )–

Promoting flowering with cold

Vernalization:Acquisition of the competence to flower in the spring by exposure to the prolonged cold of winter.

Some plants need winter to flower

No vernalization Vernalization

Plants are genetically identical

Exposed as a seedling to 4ºC for 40 days.

A role of temperature in the plant calendar

Changes in day length are a reliable indicator of seasonal progression, but day length per se is not completely informative of the time of year.

Vernalization – the process whereby flowering is promoted by a cold treatment given to a fully hydrated seed (i.e., a seed that has imbibed water) or to a growing plant.

- dry seeds do not respond to cold treatment

- without cold treatment, plants that require vernalization show delayed flowering or remain vegetative

Vernalization may involve epigenetic changes in gene expression

Requirements and features of vernalization:- requires cell division and DNA replication- requires stable changes in gene expression in meristem (even after the signal that induced the change, i.e. cold, is removed → epigenetic regulation)

Epigenetic: A heritable change in gene expression that is controlled by modifications in DNA methylation and/or chromatin structure.

- from yeast to mammals

Arabidopsis: gene acting as repressor of flowering: FLOWERING LOCUS C (FLC)

FLC - encodes MADS box transcription factor, delaying floral transition- represses transcription of AGAMOUS-LIKE 20 (AGL20)/SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), encoding MADS

box transcription factor accelerating flowering

- highly expressed in non-vernalized SAM- after vernaliztion, gene is epigenetically switched off for remainder of plant’s life cycle, permitting flowering in response to long days to occur

Vernalization blocks the expression of FLC in cold-requiring winter annual Arabidopsis ecotypes

Winter annual without cold

FLC mRNA

Winter annual after 40 days cold

Winter annual without cold, but with flc mutation

winter annual ecotypes

•VRN2: Homolog of Su(z)12

•VRN1: Myb DNA binding protein

•VIN3: PHD finger protein

•LHP1: LIKE-HETEROCHROMATIN PROTEIN

•VIL1: PHD finger protein

•VIN5: Histone arginine methyl transferase

•VIN7: PAH2 domain protein

Genes are involved in the regulation of chromatin structure

Genetic screening for vernalization insensitive (vin) mutants

Other genes involved in control of flowering by vernalization

VERNALIZATION (VRN) 1 and VRN2both are required for maintenance of low levels of FLC mRNA that are established by cold treatment once plants are exposed to warmer conditions

Role of VRN2 is to maintain the repression of FLC expression.

Time vernalized

Time vernalized

Days at 20ºC after vernalization

Days at 20ºC after vernalization

FLC mRNA

FLC mRNA

Wild type

vrn2

VRN2 encodes a gene related toDrosophila Polycomb-group (PcG) genes

In Drosophila, PcG proteins act in large protein complexes. They maintain the repression of transcription of homeotic genes, once the pattern of expression of these genes has been established during early embryo development.

VRN2Arabidopsis

SU(Z)12Drosophila

Polycomb-group complexes in Drosophila repress gene expression by modifying histones

Histone 3 is a major target for modifications – those above activate gene expression, those below repress it.

Polycomb-group proteins promote the methylation of K9 and K27 (H3K9Me, H3K9Me) – gene repression

VRN2 involved in chromatin remodeling → vernalization down-regulates FLC by epigenetic mechanisms

Histone code

Modified histone could be recognized by activation/repression complexes and establish stable activation/repression chromatin

High in:

Acetylation, H3K4Me, H3S10P

High in:

H3K9Me, H3K27Me

Active chromatin (ON)

Repressed chromatin (OFF)

Dynamics of FLC chromatin

Active FLC Chromatin

High in

Ac; H3K4Me; H3S10P

H2A.Z Histone variant

Repressed FLC Chromatin

High in

H3K9Me; H3K27Me

H4R3Me2; LHP1

VIL1, VIN5

VIN3, VIL1, VIN5 VRN2, VRN1, etc.

(from Sung and Amasino, 2005)

ON; FallWINTER!

OFF; Spring

From Lang & Melchers memory in Hyocyamus niger

Memory of winter can be mitotically stable

FLC repression by vernalization is mitotically stable

VIN3VIN3

UBQ

FLC

Molecular basis of vernalization response

•FLC is a potent repressor of flowering.

Competence: in Arabidopsis, is determined largely by FLC expression level. Vernalization leads to competence via repression of FLC.

Mitotic stability: Vernalization-mediated repression of FLC via histone modifications that are hallmarks of epigenetic silencing

No vernalization

FLC expression is subject to positive and negative regulation

Fall: flowering repressed

FLC is highly expressed and thus represses FT activationShort Days prevents CO-FT activation

FLC is repressed and FLC chromatin undergoes changes

-Stable repression of FLC by chromatin changes eliminates antagonistic effect on FT activation

- Long Days promote CO-FT activation

FLC resetting

-During meiosis, FLC is reactivated (reversing chromatin structure)

Increase in AGL20 expression during floral induction in SAM

0 h 18 h 42 h 5 d

Times after shifting the plants from short days (8h L/16 h D) to long days (16h L/8h D)

Multiple flowering control pathways

Many genes have variations even within same species

provide flexibilities to adapt to local environments

The transition to flowering involves multiple factors and pathways

The transition to flowering involves multiple factors and pathways

FTFT

The Arabidopsis calendar