1. Molecular mechanism of plant flowering time control
第十章 基因和发育
第二讲
植物开花时间控制的分子机理
Molecular mechanism of plant flowering time control
By Hongwei Guo, Peking University,

2. When to flower is a matter of the survival of species
Transition to reproduction
When to flower is a matter of the survival of species ?
Main inflorescence shoot in middle, that can branch into lateral shoot
At end of each shoot – place to make flower
Each inflorescence shoot can make leaves and lateral organs
What regulates transition from vegetative growth (main function to increase biomass of plant) to making plant sexually ready
Vegetative phase
Reproductive phase

3. 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

4. (“Maryland Mammoth” cultivar of tobacco)

8. 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)

9. Multiple grafting experiment with Perilla
Only single leaf from one of the plant treated – all 5 plants can make flower
Something made in leaf can be transferred
Flowering signal is grafting transmittable
Long distance transport---- thru vascular tissue

10. The flowering signal: florigen
vegetative or reproductive growth?
the flowering signal is generated in the leaf
the signal goes one way: from the leaf to the apex
Grafting transmittable
SAM
Florigen
Florigen can be transmitted to other plants thru grafting
Flower locus T protein claimed to be florigen – but some disputes
If you find signal or molecule that can greatly change flowering time – agriculture revolutionized
Florigen
Florigen

11. Photoperiodic flowering
Two essential questions:
1. How does the leaf measure day-length?
2. What is the florigen?

12. Genetics provides the answers
Arabidopsis: Long Day Plant
Flowering 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 receptor
PhyA: Far-red light receptor
CO: transcriptional co-activator
FT: transcriptional co-activator (?)

14. 3 wavelengths (Blue, Red, Far-red) are important in regulating plant growth and development

15. phyA also absorbs blue light
Phytochromes (光敏色素）：Red/Far-red light receptors
Cryptochromes (隐花色素）：Blue light receptors
phyA also absorbs blue light

16. Signal
Trans-duction

17. (originally proposed by Boenning, 1936)
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.

18. PRR: photoperiod response regulator

19. PRR: photoperiod response regulator

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

24. Is co the PRR that measures daylength?
LFY
A, B, C, E class genes
（春化）
Is co the PRR that measures daylength?

26. CO is required for FT expression

27. FT mRNA levels determine flowering time
LD: early flowering
SD: late flowering
phyBDE: early flowering
cry2: late flowering

33. Cry inhibits CO protein degradation in light
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.

34. Molecular mechanism of photoperiodic flowering

35. Photoperiod-dependent activation of CO protein and FT mRNA
CDF1: circadian dependent factor
CO protein measures day-length
FT mRNA level determines flowering time
CO mRNA is regulated by circadian clock.
CO protein is stabilized by light
Therefore, FT expression is activated only in long day

36. FT (or Hd3a) is a floral activator both in LD and SD plants
Long Day Plant
Short Day Plant
(Kobayashi & Weigel, 2007)
CO in Long Day plants and similar proteins (Hd1) in Short Day plants are regulated in opposite ways

38. 2005
retr

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

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

44. SAM
leaves

58. Where in the vascular tissue is FT mRNA or
FT protein locate (and migrate)?
Phloem sap: solutes migrating thru phloem (sieve tube)

64. So…….. 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

65. Photoperiodic flowering in plants
FT protein GI
FT protein
Hd3a protein

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

67. Vernalization (春化)– Promoting flowering with cold

68. Some plants need winter to flower
Vernalization:
Acquisition of the competence to flower in the spring by exposure to the prolonged cold of winter.
Plants are genetically identical
No vernalization
Vernalization
Exposed as a seedling to 4ºC for 40 days.

69. 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

70. 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

71. Vernalization blocks the expression of FLC in cold-requiring winter annual Arabidopsis ecotypes
winter annual ecotypes
Winter annual
without cold
Winter annual after 40 days cold
Winter annual without cold, but with flc mutation
FLC mRNA

72. for vernalization insensitive (vin) mutants
Genetic screening
for vernalization insensitive (vin) mutants
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
Fewer;;;

73. Other genes involved in control of flowering by vernalization
VERNALIZATION (VRN) 1 and VRN2
both are required for maintenance of low levels of FLC mRNA that are established by cold treatment once plants are exposed to warmer conditions
Wild type
Time vernalized
Days at 20ºC after vernalization
FLC mRNA
Role of VRN2 is to maintain the repression of FLC expression.
vrn2
Time vernalized
Days at 20ºC after vernalization
FLC mRNA

74. VRN2 encodes a gene related to Drosophila Polycomb-group (PcG) genes
Arabidopsis
SU(Z)12
Drosophila
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.

75. 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

76. Repressed chromatin (OFF)
Histone code
Active chromatin (ON)
Repressed chromatin (OFF)
High in:
Acetylation, H3K4Me, H3S10P
High in:
H3K9Me, H3K27Me
Modified histone could be recognized by activation/repression complexes and establish stable activation/repression chromatin

77. Dynamics of FLC chromatin
Active FLC Chromatin
High in
Ac; H3K4Me; H3S10P
H2A.Z Histone variant
Repressed FLC Chromatin
H3K9Me; H3K27Me
H4R3Me2; LHP1
VIL1, VIN5
WINTER!
VIN3, VIL1, VIN5 VRN2, VRN1, etc.
ON; Fall
OFF; Spring
(from Sung and Amasino, 2005)

78. Memory of winter can be mitotically stable
From Lang & Melchers memory in Hyocyamus niger

79. FLC repression by vernalization is mitotically stable
VIN3
VIN3
UBQ
We recapitulated fall/winter/spring seasons. Prior to vernalization as shown in red here represents fall condition, and blue indicated that plants are in the cold as in winter.

80. 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

81. No vernalization
FLC expression is subject to positive and negative regulation

83. Fall: flowering repressed
FLC is highly expressed and thus represses FT activation
Short Days prevents CO-FT activation

84. FLC is repressed and FLC chromatin undergoes changes

85. Stable repression of FLC by chromatin changes eliminates antagonistic effect on FT activation
Long Days promote CO-FT activation

86. FLC resetting During meiosis, FLC is reactivated
(reversing chromatin structure)

87. Increase in AGL20 expression during floral induction in SAM
0 h h h d
Times after shifting the plants from short days (8h L/16 h D) to long days (16h L/8h D)

88. Multiple flowering control pathways
Many genes have variations even within same species
provide flexibilities to adapt to local environments

89. The transition to flowering involves multiple factors and pathways

90. The transition to flowering involves multiple factors and pathwaysFT FT

91. The Arabidopsis calendar