Download Rhythms of Life: The Plant Circadian Clock

Rhythms of Life:
The Plant Circadian Clock
Somers, D.E. (1999). The physiology and molecular bases of the plant circadian clock. Plant Physiol. 121: 9-20.
© 2016 American Society of Plant Biologists
Living on a rotating planet is
biologically stressful
Over a 24 hour period there is
large variation in
environmental conditions
including temperature, light
intensity, humidity and
predator behavior
• Extreme day-night temperature
difference: 57 oC (-48 oC to 9 oC,
Montana, 1972)
• Typical day-night fluctuation:
~10 oC each day (central Japan)
See Kudoh, H. (2016). Molecular phenology in plants: in natura systems biology for the comprehensive understanding of seasonal responses under natural environments. New Phytol. 210: 399-412. Image: NASA.
© 2016 American Society of Plant Biologists
cycles
darkness
Active
Continuous
Inactive
LightDark
Circadian clocks are biological
oscillators with a ~24 hour period
- Higher levels of wheel running activity at night
- In continuous darkness these rhythms persist but with a
~ 23 hour period
Figure from Li, J.-D., Burton, K.J., Zhang, C., Hu, S.-B. and Zhou, Q.-Y. (2009). Vasopressin receptor V1a regulates circadian rhythms of locomotor activity
and expression of clock-controlled genes in the suprachiasmatic nuclei. Am. J. Physiol. 296: R824-R830, used with permission. Image source: Mylius.
© 2016 American Society of Plant Biologists
Circadian clocks control many
aspects of human physiology
High alertness
10.00
14.30 Best coordination
15.30 Fastest reaction time
Melatonin secretion stops
07.30
06.45
Rise in blood pressure
04.30
Lowest body temperature
17.00 Greatest muscle
strength
18.30 Highest blood
pressure
19.00 Highest body
temperature
21.00 Melatonin secretion starts
02.00
Deepest sleep
Deepest sleep
22.30 Bowel movements suppressed
Image source: Addicted04
© 2016 American Society of Plant Biologists
Plant circadian biology has a long
history
De Marian (1729) ‘Observation
botanique’ of Mimosa pudica:
Illustration of ‘sleep’ movements in
Medicago, from Charles Darwin (1880) ‘The
Power of Movement in Plants’
“sensitive to the Sun and daylight:
the leaves & their peduncles fold
themselves away & contract around
sunset, in the same way they do
when the Plant is touched or
shaken.”
Image sources: H. Zell, Charles Darwin “Power of Movement in Plants”
© 2016 American Society of Plant Biologists
Architecture of the circadian clock
© 2016 American Society of Plant Biologists
Principles of operation of plant
circadian clocks
Aspect of the Circadian
System
Biological Function
Circadian oscillator
Generate a rhythm with a ~24h period within the
cell
Entrainment pathways
Synchronize the oscillator with the external time
of day so that the clock stays accurate
Output pathways
Communicate temporal information from the
oscillator to other parts of the cell
Circadian gating
Adjust the sensitivity of entrainment and output
pathways depending on the time of day
© 2016 American Society of Plant Biologists
Interconnected parts of the
circadian system
Environmental
Inputs
Circadian gating of
entrainment and outputs
Gene
Output
pathways
Entrainment
pathways
Circadian
oscillator
Rhythms in:
- transcription
- physiology
- biochemistry
© 2016 American Society of Plant Biologists
The circadian oscillator
Most circadian clocks are
transcription-translation feedback loops
The protein encoded by
Gene A activates Gene B
Protein A
Gene A
The protein encoded by
Gene B represses Gene A
Gene B
Protein B
© 2016 American Society of Plant Biologists
The circadian oscillator
Gene A
Gene B
Protein B
Gene transcript abundance
Protein A
The feedback loop results in
rhythms of transcript abundance
of the two genes
Gene A
Gene B
0
12
24
36
Time (hours)
• Reciprocal feedback loop
• Negative feedback step
• Speed of biochemical reactions adds a rate constant
48
Simple biological
oscillator
© 2016 American Society of Plant Biologists
An early model for the functioning of
the circadian clock in Arabidopsis
• This is one of the first structures
proposed for the plant circadian
clock
Protein A
Activation
(out of date!)
Gene B
Suppression
Gene A
Protein B
• It is an oscillator with activation
and suppression feedback
(compare with previous slide)
• The main genes involved are
TOC1, LHY and CCA1
• The model is out of date (TOC1
actually suppresses CCA1), but
provides an example of
oscillator structure
From Alabadı́, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Más, P. and Kay, S.A. (2001). Reciprocal regulation between
TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science. 293: 880-883. Reprinted with permission from AAAS
© 2016 American Society of Plant Biologists
Reciprocal repression between CCA1
and TOC1 at the core of the circadian
clock
Overexpression of CCA1 suppresses
circadian oscillations of TOC1
CCA1 and LHY bind to the promoter
of TOC1
Overexpression of TOC1 suppresses
circadian oscillations of CCA1
The CCT domain of TOC1 is required
for it to bind the CCA1 promoter
From Alabadı́, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Más, P. and Kay, S.A. (2001). Reciprocal regulation between TOC1 and LHY/CCA1 within the
Arabidopsis circadian clock. Science. 293: 880-883. Reprinted with permission from AAAS. Gendron, J.M., Pruneda-Paz, J.L., Doherty, C.J., Gross, A.M., Kang,
S.E. and Kay, S.A. (2012). Arabidopsis circadian clock protein, TOC1, is a DNA-binding transcription factor. Proc. Natl. Acad. Sci. USA 109: 3167-3172.
© 2016 American Society of Plant Biologists
The current model of the circadian
oscillator is a complex network
Note TOC1 is a
suppressor of
CCA1/LHY
(compare with
previous model)
Morning Loop
Evening Complex
Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: 240-249 with permission from Elsevier..
© 2016 American Society of Plant Biologists
Different clock
components
are expressed
at different
times of day
•
Network models indicate
connections between
components, but lack temporal
information about clock
function
This example shows how three
clock genes are activated at
different times of day/night
5
Normalised transcript abundance
•
4
3
2
CCA1
1
PRR9
LUX
0
-1
0
4
8
12
16
Time (h)
20
24
Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: 240-249 with permission from Elsevier.. Data from DIURNAL database: http://diurnal.mocklerlab.org/
© 2016 American Society of Plant Biologists
The circadian clock also includes
post-transcriptional processes
Chromatin remodeling:
-e.g. promoter of TOC1 has a clock-controlled pattern of
histone 3 (H3) acetylation that affects TOC1 expression
Control of protein stability by proteasome:
-e.g. ZTL is involved in dark-dependent degradation of the
TOC1 protein.
Phosphorylation:
-e.g. Casein Kinase 2 (CK2) phosphorylates CCA1 and LHY
Cytosolic signaling molecules:
-e.g. circadian rhythms of cADPR and Ca2+ in the cytosol
regulate the dynamics of the oscillator
See Más, P. (2008) Circadian clock function in Arabidopsis thaliana: time beyond transcription. Trends Cell Biol. 18: 273-281 for review
© 2016 American Society of Plant Biologists
The circadian oscillator is
temperature-compensated
Temperature (oC)
Col-0 wild type
maintains a ~24 period
across a range of
temperatures, i.e. is
temperature
compensated
Period (h)
PRR7/PRR9
knockdown changes
period in response to
temperature change
22 oC
28 oC
Period (h)
PRR7, PRR9 and FBH1 have roles
in temperature compensation
Wild type
FBH1-ox
FBH1-ox
Salomé, P.A., Weigel, D. and McClung, C.R. (2010). The role of the Arabidopsis Morning loop components CCA1, LHY, PRR7, and PRR9 in temperature compensation. Plant Cell. 22: 3650-3661;
Nagel, D.H., Pruneda-Paz, J.L. and Kay, S.A. (2014). FBH1 affects warm temperature responses in the Arabidopsis circadian clock. Proc. Natl. Acad. Sci. USA 111: 14595-14600.
© 2016 American Society of Plant Biologists
Why is entrainment required?
The time of dawn and dusk is different every single day
Time of day (24 h clock)
08:00
06:00
Time of sunrise
04:00
02:00
0
100
200
300
400
20:00
18:00
Time of sunset
16:00
14:00
0
100
200
300
400
Location: Bristol, UK
51.4500° N, 2.5833° W
Day of year
© 2016 American Society of Plant Biologists
Why is entrainment required?
• Measurements of
circadian period in
three wild type strains
of Arabidopsis thaliana
• There is variation
within and between
strains, but all have a
period of approximately
24 hours
Measurements
The period of the circadian oscillator is approximately
24 h and there is natural variation between plants
Circadian period (h)
Reprinted from Swarup, K., Alonso-Blanco, C., Lynn, J.R., Michaels, S.D., Amasino, R.M., Koornneef, M. and Millar, A.J. (1999). Natural allelic variation identifies new genes in the Arabidopsis circadian system. Plant J. 20: 67-77.
© 2016 American Society of Plant Biologists
Several environmental signals
entrain the circadian oscillator
Red light
(phytochrome photoreceptors)
Blue light
(cryptochrome photoreceptors)
Sugars produced by photosynthesis
Temperature fluctuations
Circadian
oscillator
© 2016 American Society of Plant Biologists
Phytochromes and cryptochromes
provide light input to the circadian
clock
phyA mutant
Wild type
phyB mutant
Wild type
Fluence rate of red light
(µmol m-2 s-1)
cry1 mutant
Wild type
cry2 mutant
Wild type
Fluence rate of blue light
(µmol m-2 s-1)
From Somers, D.E., Devlin, P.F. and Kay, S.A. (1998). Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock. Science. 282: 1488-1490 Reprinted with permission from AAAS
© 2016 American Society of Plant Biologists
Circadian clocks regulate plant cells
by controlling gene expression
• Some circadian clock proteins are transcription factors
that regulate sets of genes with a circadian rhythm
Example: a
daytime
transcription
factor
Day
TF
TF
Gene 1
TF
Gene 2
TF
Gene 3
Night
TF
Gene 1
Gene 2
Gene 3
© 2016 American Society of Plant Biologists
Specific gene promoter sequences
may underlie specific circadian
phases of transcription
• These cis elements occur
with high frequency in
promoters of transcript sets
with certain circadian
phases
• Indicates that the circadian
clock regulates different
subsets of genes with
different circadian phases
through particular clockcontrolled promoter motifs
Covington, M.F., Maloof, J.N., Straume, M., Kay, S.A. and Harmer, S.L. (2008). Global transcriptome analysis
reveals circadian regulation of key pathways in plant growth and development. Genome Biology. 9: 1-18.
© 2016 American Society of Plant Biologists
The importance of circadian rhythms
in plant biology
© 2016 American Society of Plant Biologists
Plants with a functioning circadian
clock that matches the environment
grow larger
(wildtype)
Col-0 = wildtype
CCA1-ox = arrhythmic transgenic line
T24 = 12 h light, 12 h dark
T20 = 10 h light, 10 h dark
T24 = 12 h light, 12 h dark
T28 = 14 h light, 14 h dark
From Dodd, A.N., Salathia, N., Hall, A., Kévei, E., Tóth, R., Nagy, F., Hibberd, J.M., Millar, A.J. and Webb, A.A.R. (2005). Plant circadian clocks
increase photosynthesis, growth, survival, and competitive advantage. Science. 309: 630-633. Reprinted with permission from AAAS.
© 2016 American Society of Plant Biologists
Relative transcript abundance
The circadian clock controls
multiple aspects of plant biology
Molecular Biology:
Physiology:
~30% of the Arabidopsis
thaliana transcriptome oscillates
with a 24 period
Stomatal opening and closing are
under the control of the circadian
oscillator
Circadian
rhythms of gas
exchange
= wildtype
= arrhythmic
mutant (CCA1ox)
Time (h)
From Harmer, S.L., Hogenesch, J.B., Straume, M., Chang, H.-S., Han, B., Zhu, T., Wang, X., Kreps, J.A. and Kay, S.A. (2000). Orchestrated transcription of key pathways in
Arabidopsis by the circadian clock. Science. 290: 2110-2113 and from Dodd, A.N., Salathia, N., Hall, A., Kévei, E., Tóth, R., Nagy, F., Hibberd, J.M., Millar, A.J. and Webb,
A.A.R. (2005). Plant circadian clocks increase photosynthesis, growth, survival, and competitive advantage. Science. 309: 630-633. Reprinted with permission from AAAS.
© 2016 American Society of Plant Biologists
The circadian clock controls
multiple aspects of plant biology
Growth:
Development:
Hypocotyl elongation is clock
controlled
Photoperiod is one of the environmental
factors controlling flowering
Plants grown under long days:
Wildtype
Circadian clock
mutant (gigantea)
Reprinted with permission from from Dowson-Day, M.J. and Millar, A.J. (1999). Circadian dysfunction causes aberrant hypocotyl elongation
patterns in Arabidopsis. Plant J. 17: 63-71 and Amasino, R. (2010). Seasonal and developmental timing of flowering. Plant J. 61: 1001-1013.
© 2016 American Society of Plant Biologists
The circadian clock gives plants a
fitness advantage
Arabidopsis thaliana mutant lines
with endogenous circadian period:
Endogenous
period
Environment
(= 20 h)
(= 28 h)
~20 h
~28 h
Competition experiments:
When the endogenous period
matches the external light-dark
cycles, plants perform better in
terms of:
• survival
• biomass (dry and fresh
weight)
• chlorophyll content
From Dodd, A.N., Salathia, N., Hall, A., Kévei, E., Tóth, R., Nagy, F., Hibberd, J.M., Millar, A.J. and Webb, A.A.R. (2005). Plant circadian clocks
increase photosynthesis, growth, survival, and competitive advantage. Science. 309: 630-633. Reprinted with permission from AAAS.
© 2016 American Society of Plant Biologists
Investigating the circadian clock in
the laboratory
© 2016 American Society of Plant Biologists
Time-course analysis is used to
study circadian rhythms in plants
The plant is first grown in
cycles of light and dark
Biological
process
The plant is then transferred to
conditions of constant light (or dark) and
temperature, where circadian-regulated
biological process will ‘free run’
Light Dark Light Dark
0
12
24
36
48
Time (h)
0
12
24
36
48
60
Time (h)
72
84
96
The time that would have been dark is referred to as ‘subjective night’, and is
sometimes indicated by grey bars on circadian time-courses
© 2016 American Society of Plant Biologists
Circadian rhythms have a number of
measurable properties
Biological process
‘Subjective dawn’ occurs
every 24h after lights on
Phase = time of peak relative to
subjective dawn
Period
Amplitude
0
12
24
36
48
60
72
84
96
Time (h)
Period = time to complete one full cycle
Phase = the time at which a particular point of cycle occurs (e.g. the peak)
Amplitude = the displacement of the oscillation from the center point
© 2016 American Society of Plant Biologists
Common methods for studying circadian
rhythms: Transcript analysis
Individual transcripts
can be monitored
or
Circadian rhythms of the whole
transcriptome can be studied with
microarrays or RNA sequencing
Light harvesting complex
transcripts
Photosystem I and II
transcripts
This transcriptome
analysis found that
many photosynthesis
genes have circadian
rhythms
From Harmer, S.L., Hogenesch, J.B., Straume, M., Chang, H.-S., Han, B., Zhu, T., Wang, X., Kreps, J.A. and Kay, S.A. (2000). Orchestrated
transcription of key pathways in Arabidopsis by the circadian clock. Science. 290: 2110-2113. Reprinted with permission from AAAS.
© 2016 American Society of Plant Biologists
Non-invasive measurement
techniques benefit the study of
circadian rhythms
• Measurements of a biological property need to be made
frequently (e.g., hourly) over several days
• Destructive sampling to obtain RNA or protein is
inconvenient:
• Substantial quantities of plant material required, long
working hours, opportunities for human error
• Non-invasive and automated measurement techniques
have been developed
• Destructive sampling is sometimes essential to monitor
rhythms of transcripts, proteins or metabolites
© 2016 American Society of Plant Biologists
In Arabidopsis, leaf
movements are
part of rhythmic
patterns in growth
9 days old 8 days old 8 days old
(subj. day) (subj. night) (subj. day)
Some plants have a
pulvinus at the base
of the leaf which
drives movement of
the leaves
Leaf position (pixels)
Common methods for studying
circadian rhythms: Leaf movement
0
24
48
72
96
120
144
Automated video imaging and
image analysis allows
quantification of leaf movements
From Hicks, K.A., Millar, A.J., Carré, I.A., Somers, D.E., Straume, M., Meeks-Wagner, D.R. and Kay, S.A. (1996). Conditional circadian dysfunction of the
Arabidopsis early-flowering 3 mutant. Science. 274: 790-792. Reprinted with permission from AAAS. Image credits: Vojtěch Zavadil; K.Hubbard unpublished
© 2016 American Society of Plant Biologists
Common methods for studying
circadian rhythms:
Bioluminescence imaging
Expression in plants of an enzyme from fireflies called
luciferase causes plants to emit light when provided with the
substrate luciferin
Light
O2
Luciferin
ATP
Plant genome
LUCIFERASE
Oxyluciferin
AMP
LUCIFERASE transgene
© 2016 American Society of Plant Biologists
Common methods for studying
circadian rhythms:
Bioluminescence imaging
•Placing LUCIFERASE
under the control of a
promoter with a circadian
rhythm allows the rhythm to
be monitored.
Luciferase bioluminescence
imaged from Arabidopsis
seedlings
•The plant emits circadian
rhythms of light that can be
detected with a very
sensitive camera.
Millar, A.J., Short, S.R., Chua, N.H. and Kay, S.A. (1992). A novel circadian phenotype based on firefly luciferase expression in transgenic plants. Plant Cell. 4: 1075-1087.
© 2016 American Society of Plant Biologists
Common methods for studying
circadian rhythms:
Bioluminescence imaging
• LUCIFERASE is placed
under the control of the
rhythmic CAB2 promoter
Mutant with short
circadian period (toc1-1)
Wild type
seedlings
• Around 8000 seedlings
from a mutagenized
population were tested, to
identify components of the
circadian clock
• This allowed identification
of circadian clock
components (here, TOC1)
From Millar, A., Carre, I., Strayer, C., Chua, N. and Kay, S. (1995). Circadian clock mutants in Arabidopsis identified by luciferase imaging. Science. 267: 1161-1163. Reprinted with permission from AAAS.
© 2016 American Society of Plant Biologists
Advanced methods for studying
circadian rhythms:
Rhythms in individual tissues
Bioluminescence from
individual pixels
1. Use a super sensitive camera with close up lens to monitor luciferase
Luciferase bioluminescence
from single Arabidopsis leaf
Wenden, B., Toner, D.L.K., Hodge, S.K., Grima, R. and Millar, A.J. (2012). Spontaneous spatiotemporal waves of gene expression from biological clocks in the leaf. Proc. Natl. Acad. Sci. USA. 109: 6757-6762.
© 2016 American Society of Plant Biologists
Advanced methods for studying
circadian rhythms:
Rhythms in individual tissues
2. Split luciferase into two fragments, and give
one a tissue-specific gene promoter
This half of luciferase is only
expressed when the clock promoter
is active
The complete protein is only
assembled when both
promoters are active
This half of luciferase is only
expressed in the specific tissue type
Reprinted by permission from Macmillan Publishers Ltd: Endo, M., Shimizu, H., Nohales, M.A., Araki, T. and Kay, S.A. (2014). Tissue-specific clocks in Arabidopsis show asymmetric coupling. Nature. 515: 419-422.
© 2016 American Society of Plant Biologists
Advanced methods for studying
circadian rhythms:
Rhythms in individual tissues
2. Split luciferase into two fragments, and give
one a tissue-specific gene promoter
One half of luciferase: Vascular SUC2 promoter
One half of luciferase: Circadian TOC1 promoter
1 mm
This was used to show the circadian clock
in vascular tissue is dominant to that of
other cell types in leaves
Reprinted by permission from Macmillan Publishers Ltd: Endo, M., Shimizu, H., Nohales, M.A., Araki, T. and Kay, S.A. (2014). Tissue-specific clocks in Arabidopsis show asymmetric coupling. Nature. 515: 419-422.
© 2016 American Society of Plant Biologists
Advanced methods for studying
circadian rhythms: The value of
mathematical modeling
How do you unravel how this functions? It’s totally non-intuitive!
Large number of interconnected clock components
Multiple feedback loops, both positive and negative
Each protein and transcript has its own unique rate of synthesis and breakdown
Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: 240-249 with permission from Elsevier..
© 2016 American Society of Plant Biologists
Advanced methods for studying
circadian rhythms: The value of
mathematical modeling
Building mathematical simulations of the clock has:
• Provided new information about how clock
components interact
• Provided explanations for why the clock is so complex
• Provided new information about how the clock is
entrained to the environment
• Demonstrated the power of mathematical and
systems biology approaches for biological research
© 2016 American Society of Plant Biologists
The circadian clock and plant
metabolism
© 2016 American Society of Plant Biologists
The circadian clock has extensive
control of plant metabolism
Almost all metabolic
pathways include at
least one enzyme that is
under circadian
transcriptional control
•
•
Each square = One circadianregulated transcript
Color of square = phase of
expression
Reprinted from Farré, E.M. and Weise, S.E. (2012). The interactions between the circadian clock and primary metabolism. Curr. Opin. Plant Biol. 15: 293-300 with permission from Elsevier.
© 2016 American Society of Plant Biologists
Many metabolic pathways have
physiologically appropriate phases
of maximal transcript abundance
Subjective time of day:
Dawn
Relative Expression Level
1.6
Dawn
Dusk
Dawn
1.4
Chlorophyll biosynthesis
genes peak just before dawn
to anticipate light availability
1.2
1
0.8
0.6
Chlorophyll Biosynthesis
0.42
Relative Expression Level
Dusk
0
1.5
12
24
36
Time in continuous light (h)
48
Starch catabolism genes peak
around dusk
1
0.5
Starch Catabolism
0
0
12
24
36
Time in continuous light (h)
48
Data from DIURNAL database: http://diurnal.mocklerlab.org/
© 2016 American Society of Plant Biologists
Circadian clock mutants have
altered metabolite levels in lightdark cycles
Data shown for a prr9/7/5
triple mutant grown in
12h light: 12h dark cycles
Increase in Shikimate
suggests changes in
secondary metabolism
Increase in Citric Acid
Cycle intermediates
Fukushima, A., Kusano, M., Nakamichi, N., Kobayashi, M., Hayashi, N., Sakakibara, H., Mizuno, T. and Saito, K. (2009). Impact of
clock-associated Arabidopsis pseudo-response regulators in metabolic coordination. Proc. Natl. Acad. Sci. USA 106: 7251-7256.
© 2016 American Society of Plant Biologists
Carbohydrate degradation at night is
temporally controlled
The rate of starch
degradation is related to
the length of the night, so
that the plant only exhausts
starch reserves just before
the end of the night
12hL:12hD cycles (normal growth condition)
Early night imposed
Graf, A., Schlereth, A., Stitt, M. and Smith, A.M. (2010). Circadian control of carbohydrate availability for growth in Arabidopsis plants at night. Proc. Natl. Acad. Sci. USA 107: 9458-9463.
© 2016 American Society of Plant Biologists
cca1/lhy mutants exhaust starch
reserves at night
Relative Expression Level
cca1/lhy mutants:
-Accumulate 20% less
starch than wildtypes
-Degrade starch 35% faster
than wildtypes at night
-Exhaust starch reserves 34 hours before the end of
the night
-Express starvation genes
before the end of the night
wildtype
cca1-11/lhy-21
Graf, A., Schlereth, A., Stitt, M. and Smith, A.M. (2010). Circadian control of carbohydrate availability for growth in Arabidopsis plants at night. Proc. Natl. Acad. Sci. USA 107: 9458-9463.
© 2016 American Society of Plant Biologists
The chloroplast has circadian
rhythms of gene expression that are
controlled by the nucleus
psbD = Chloroplast gene
Circadian
oscillator
TF
SIG5
SIG5 imported
into chloroplast
SIG5
SIG5 = Nuclear gene
cytosol
Gene
chloroplast
chloroplast
From Noordally, Z.B., Ishii, K., Atkins, K.A., Wetherill, S.J., Kusakina, J., Walton, E.J., Kato, M., Azuma, M., Tanaka, K., Hanaoka, M. and Dodd, A.N.
(2013). Circadian control of chloroplast transcription by a nuclear-encoded timing signal. Science. 339: 1316-1319. Reprinted with permission from AAAS.
© 2016 American Society of Plant Biologists
Primary metabolites regulate the
activity of the circadian oscillator
Application of external
sucrose restores circadian
rhythms in wildtype
seedlings grown in the dark
This is dependent on the
presence of the oscillator
component GIGANTEA
Dalchau, N., Baek, S.J., Briggs, H.M., Robertson, F.C., Dodd, A.N., Gardner, M.J., Stancombe, M.A., Haydon, M.J., Stan, G.-B., Gonçalves, J.M. and Webb, A.A.R. (2011). The
circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis thaliana circadian clock to sucrose. Proc. Natl. Acad. Sci. USA 108: 5104-5109.
© 2016 American Society of Plant Biologists
The oscillator, environmental
signalling and metabolism form an
integrated network
Environmental
Signalling
Light
Temperature
Central Oscillator
CCA1
PRR7/5/9
GI
TOC1
Metabolism
NAD+
Chloroplasts
Photosynthesis
Sugar
Redox
ATP/NAD+
Mitochondria
Redox
ATP/NAD+
Image based on Farré, E.M. and Weise, S.E. (2012). The interactions between the circadian clock and primary metabolism. Curr. Opin. Plant Biol. 15: 293-300 and
Haydon, M.J., Hearn, T.J., Bell, L.J., Hannah, M.A. and Webb, A.A.R. (2013). Metabolic regulation of circadian clocks. Semin. Cell Devel. Biol. 24: 414-421.
© 2016 American Society of Plant Biologists
The circadian clock regulates
production of volatile compounds
Petunias use the emission
of volatile compounds to
attract nocturnal pollinators
such as hawkmoths
μg emitted/g fresh weight/hr
There are circadian rhythms of
volatile emissions:
Fenske, M.P., Hewett Hazelton, K.D., Hempton, A.K., Shim, J.S., Yamamoto, B.M., Riffell, J.A. and Imaizumi, T. (2015). Circadian clock gene
LATE ELONGATED HYPOCOTYL directly regulates the timing of floral scent emission in Petunia. Proc. Natl. Acad. Sci. USA 112: 9775-9780.
© 2016 American Society of Plant Biologists
The circadian clock regulates
production of volatile compounds
Many genes required for volatile production are under transcriptional
control of the oscillator:
Circadian
transcriptional
control
Volatile
compounds
Fenske, M.P., Hewett Hazelton, K.D., Hempton, A.K., Shim, J.S., Yamamoto, B.M., Riffell, J.A. and Imaizumi, T. (2015). Circadian clock gene
LATE ELONGATED HYPOCOTYL directly regulates the timing of floral scent emission in Petunia. Proc. Natl. Acad. Sci. USA 112: 9775-9780.
© 2016 American Society of Plant Biologists
Circadian mutants of Petunia do not
emit volatile compounds at night
Transgenic overexpression of PvLHY
abolishes nocturnal production of
volatiles
W115 = wildtype line
#37* = PvLHY overexpressing line
Fenske, M.P., Hewett Hazelton, K.D., Hempton, A.K., Shim, J.S., Yamamoto, B.M., Riffell, J.A. and Imaizumi, T. (2015). Circadian clock gene
LATE ELONGATED HYPOCOTYL directly regulates the timing of floral scent emission in Petunia. Proc. Natl. Acad. Sci. USA 112: 9775-9780.
© 2016 American Society of Plant Biologists
Plants are more resistant to herbivory
when their circadian rhythms are
phased with rhythms of insects
When In Phase the plants
resist herbivore attack
Insects
Plants
Insects
Plants
Entrainment
conditions
Free run
(constant dark)
When Out of Phase the plants
are vulnerable to herbivores
Goodspeed, D., Chehab, E.W., Min-Venditti, A., Braam, J. and Covington, M.F. (2012). Arabidopsis synchronizes jasmonate-mediated defense with insect circadian behavior. Proc. Natl. Acad. Sci. USA 109: 4674-4677.
© 2016 American Society of Plant Biologists
Plants produce jasmonates during
the subjective day to deter
herbivores
Insects feed during
the subjective day
Jasmonates accumulate
during the subjective day
Jasmonate accumulation induces herbivore defense mechanisms
e.g. production of toxic secondary metabolites
Goodspeed, D., Chehab, E.W., Min-Venditti, A., Braam, J. and Covington, M.F. (2012). Arabidopsis synchronizes jasmonate-mediated defense with insect circadian behavior. Proc. Natl. Acad. Sci. USA 109: 4674-4677.
© 2016 American Society of Plant Biologists
The circadian clock provides timing
information to control photoperiodic
flowering
© 2016 American Society of Plant Biologists
Many plants use photoperiod as a
signal to sense seasonal changes
Summer = long
photoperiod
(~15 h of light)
Winter = short
photoperiod
(~7 h of light)
16
14
12
10
8
Cambridge, UK
6
Aug
Jul
Jun
May
Apr
Mar
Feb
Jan
Dec
Nov
Oct
4
Sep
Day length (hours)
18
Wheat (Triticum
aestivum) flowers in the
spring when the days
are getting longer
Image source: anthere
© 2016 American Society of Plant Biologists
Photoperiod sensitive plants induce
flowering under either long or short
days
Long Day Plants
e.g., Wheat
24 hours
Flash
Critical night length
Short Day Plants
e.g., Rice
24 hours
Flash
Critical night length
(Note: flowering of some species e.g. tomato is photoperiod insensitive; these are referred to as day-neutral plants)
© 2016 American Society of Plant Biologists
Flowering time is a highly regulated
event which centers on FLOWERING
LOCUS T (FT)
Photoperiod
Age
(autonomous
pathway)
Gibberellin
Cold winters
(vernalization
pathway)
Induction of FT
expression in leaves
FT protein
moves to the
shoot apical
meristem via
the phloem
Floral integrator
genes induced
in meristem, and
flowering is
initiated
© 2016 American Society of Plant Biologists
The external coincidence model
explains photoperiodic induction of
flowering time in long days
= CONSTANS
= FT
First proposed by Bünning (1936). Model redrawn from Imaizumi, T. and Kay, S.A. Photoperiodic control of flowering: not only by coincidence. Trends Plant Sci. 11: 550-558.
© 2016 American Society of Plant Biologists
The zinc finger transcription factor
CONSTANS is the circadian
dependent regulator
50
No. of leaves at flowering
45
40
35
30
25
WT (Ler)
20
Zn
ion
co-2
15
10
5
0
Long Days
(16hL: 8hD)
Short Days
(8hL: 16hD)
CONSTANS is required for
floral induction in long days
in Arabidopsis
The zinc finger is a DNA
interaction motif found in all
kingdoms of life
Data: K. Hubbard (unpublished). Image source: Thomas Splettstoesser
© 2016 American Society of Plant Biologists
A molecular model to explain
photoperiodic control of flowering
time in Arabidopsis
The expression of
CO is controlled
by the circadian
clock, with peak
expression ~12
hours after dawn
CO protein is
unstable in the
dark due to
COP1 activity so
it doesn’t
accumulate and
FT is not induced
In long days the
peak of CO mRNA
is during the light,
so the CO protein
can accumulate
CO induces FT
expression, which
stimulates the
floral transition
Model redrawn from Imaizumi, T. and Kay, S.A. Photoperiodic control of
flowering: not only by coincidence. Trends Plant Sci. 11: 550-558.
© 2016 American Society of Plant Biologists
The photoperiodic flowering
pathway is broadly conserved
between Arabidopsis and crops
Cockram, J., Jones, H., Leigh, F.J., O'Sullivan, D., Powell, W., Laurie, D.A. and Greenland, A.J. (2007). Control of flowering time in temperate
cereals: genes, domestication, and sustainable productivity. J Exp. Bot. 58: 1231-1244 by permission of Oxford University Press.
© 2016 American Society of Plant Biologists
A small change to the model also
explains flowering in short day
plants such as rice
Long Day Plants:
CO activates FT expression
Short Day Plants:
‘CO’ represses ‘FT’ expression
Model redrawn from Song, Y.H., Shim, J.S., Kinmonth-Schultz, H.A. and Imaizumi, T. (2015). Photoperiodic flowering: Time measurement mechanisms in leaves. Annu. Rev. Plant Biol. 66: 441-464.
© 2016 American Society of Plant Biologists
Circadian gating
© 2016 American Society of Plant Biologists
Circadian gating: General concept
• The regulation of other cell signaling pathways by
the circadian clock is known as circadian gating
• It is a fundamental way that circadian clocks
regulate plant cells
• During gating, the clock acts as a valve on the
response of the plant to the environment, so the
same environmental cue causes a different strength
response depending on the time of day
• At some times of day the gate is open and the
signal passes through. At other times of day the
gate is closed and the signal cannot pass through
© 2016 American Society of Plant Biologists
Circadian gating: General concept
General principle: an example of light signaling
Response
Gate
open
Strong response
to light
Identical
light
stimulus
Gate
closed
Very weak
response to
light
© 2016 American Society of Plant Biologists
Circadian gating acts upon circadian
entrainment and environmental
signaling pathways
1. The circadian clock gates the light and
temperature signals that entrain the
circadian clock
2. The circadian clock gates signaling
pathways that regulate the responses of
plants to the environment
© 2016 American Society of Plant Biologists
1. Circadian gating of light input to
the circadian clock
• If the circadian clock
responded identically to
light at every time of day, it
would be reset to dawn
continuously and be unable
to provide a measure of
time
• The clock regulates its own
sensitivity to light, so the
way it responds to light
depends on the time of day
Red light
Blue light
Figure reprinted from Hotta, C.T., Gardner, M.J., Hubbard, K.E., Baek, S.J., Dalchau, N., Suhita, D., Dodd, A.N. and Webb, A.A.R. (2007). Modulation of environmental responses of plants by circadian clocks. Plant Cell
Environ. 30: 333-349, redrawn from Covington, M.F., Panda, S., Liu, X.L., Strayer, C.A., Wagner, D.R. and Kay, S.A. (2001). ELF3 modulates resetting of the circadian clock in Arabidopsis. Plant Cell. 13: 1305-1316.
© 2016 American Society of Plant Biologists
2. Circadian gating of environmental
response pathways
Example: The circadian clock gates responses of plants to a cold environment
***
----
----
***
***
----
***
***
CBF2 transcript
level
***
***
More sensitive to cold when transferred to cold at this time
---- Less sensitive to cold when transferred to cold at this time
Fowler, S.G., Cook, D. and Thomashow, M.F. (2005). Low temperature induction of Arabidopsis CBF1, 2, and 3 is gated by the circadian clock. Plant Physiol. 137: 961-968.
© 2016 American Society of Plant Biologists
3. Circadian gating of environmental
response pathways
Change in hypocotyl length (%)
Example: The circadian clock gates shade avoidance in Arabidopsis
Hypocotyl
• Seedling hypocotyls elongate rapidly in
the shade to overtop their competitors
Time in constant light (h)
• Seedlings are most sensitive to
simulated shade around subjective dusk
Reprinted by permission from Macmillan Publishers Ltd: Salter, M.G., Franklin, K.A. and Whitelam, G.C. (2003). Gating of the rapid shade-avoidance response by the circadian clock in plants. Nature. 426: 680-683.
© 2016 American Society of Plant Biologists
The potential for crop improvement
using circadian-dependent traits
© 2016 American Society of Plant Biologists
Circadian clock genes are
associated with agronomic traits
Species
QTL/locus
Gene in
species
Arabidopsis
homologue
Role/Trait
Eudicots
P. sativum
HR/QTL3
HR
ELF3
Monocots
O. sativa
-
OsPRR1
TOC1
-
OsPRR37
PRR3/PRR7
Ef7/hd17
OsELF3-1
ELF3
H. vulgare
Ppd-H1
HvPRR37
PRR3/PRR7
Flowering time
T. aestivum
-
Ppd-D1
PRR3/7
Flowering time
Z. mays
-
ZmGI1
GI
Circadian clock function,
flowering time, light response
Flowering time
Light-dependent circadian clock
regulation
Flowering time and growth
regulation
Table based on Bendix, C., Marshall, Carine M. and Harmon, Frank G. (2015). Circadian clock genes universally control key agricultural traits. Mol. Plant. 8: 1135-1152.
© 2016 American Society of Plant Biologists
Case study 1: A slower clock was
selected for during the
domestication of tomato
• Two QTLs were identified,
one of which mapped to a
homologue of an
Arabidopsis light signaling
protein (EID1)
• Plants with the phase delay
mutation were late
flowering and had higher
chlorophyll content in long
days, indicating a
competitive advantage
Reprinted by permission from Macmillan Publishers Ltd: Muller, N.A., Wijnen, C.L., Srinivasan, A., Ryngajllo, M., Ofner, I., Lin, T., Ranjan, A., West, D., Maloof, J.N., Sinha,
N.R., Huang, S., Zamir, D. and Jimenez-Gomez, J.M. (2016). Domestication selected for deceleration of the circadian clock in cultivated tomato. Nat Genet. 48: 89-93.
© 2016 American Society of Plant Biologists
Case study 2: The role of Ppd-H1 in
photoperiodic responses of barley
Barley (Hordeum vulgare):
- Long-day plant, i.e., it requires
day lengths in excess of a critical
minimum to flower.
- There are also varieties that are
insensitive to day length
Photoperiod
sensitive
‘Igri’
x
Photoperiod
insensitive
‘Triumph’
Mapping
population
generated
Ppd-H1 locus identified
Image source: Craig Nagy
© 2016 American Society of Plant Biologists
Ppd-H1 is a homologue of the
Arabidopsis PRR7 oscillator gene
Phenotypes of
homozygous Ppd-H1 (left),
heterozygous (middle), and
homozygous ppd-H1 (right) plants
At PRR7 is a pseudo-response
regulator that has 50% overall
similarity to Hv Ppd-H1
From Turner, A., Beales, J., Faure, S., Dunford, R.P. and Laurie, D.A. (2005). The Pseudo-Response Regulator Ppd-H1 provides adaptation to photoperiod in barley. Science. 310: 1031-1034. Reprinted
with permission from AAAS. Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: 240-249 with permission from Elsevier..
© 2016 American Society of Plant Biologists
Day length (hours)
Winter barley (Ppd-H1) is
photoperiod sensitive and flowers in
early spring
18
16
14
12
10
8
6
4
Cambridge, UK
Lebanon
Autumn
Sep
Oct
Winter
Nov
Dec
Winter barley (Ppd-H1)
Sowing
Jan
Spring
Feb
Mar
Apr
Summer
May
Jun
Jul
Aug
Flowering is induced when days get
longer than ~13 hours (i.e. in the spring),
resulting in early harvest
Harvest
Harvest occurs
before heat of
summer
See Cockram, J., Jones, H., Leigh, F.J., O'Sullivan, D., Powell, W., Laurie, D.A. and Greenland, A.J. (2007). Control of
flowering time in temperate cereals: genes, domestication, and sustainable productivity. J Exp. Bot. 58: 1231-1244.
© 2016 American Society of Plant Biologists
Day length (hours)
Spring barley (ppd-H1) is
photoperiod insensitive and flowers
late
18
16
14
12
10
8
6
4
Cambridge, UK
Lebanon
Autumn
Sep
Oct
Winter
Nov
Dec
Jan
Spring
Feb
Mar
Apr
Summer
May
Jun
Jul
Aug
Flowering fails to be induced when
days get longer than ~13 hours (i.e.,
in the spring), therefore crop grows
throughout the summer
Spring barley (ppd-H1)
Sowing in
spring avoids
frost damage
Sowing
Harvest
See Cockram, J., Jones, H., Leigh, F.J., O'Sullivan, D., Powell, W., Laurie, D.A. and Greenland, A.J. (2007). Control of
flowering time in temperate cereals: genes, domestication, and sustainable productivity. J Exp. Bot. 58: 1231-1244.
© 2016 American Society of Plant Biologists
Photoperiod insensitive landraces of
barley are more common in northern
Europe
Autumn
Winter
Spring
Summer
North = spring barley (ppd-H1)
Sowing
Spread of barley
northwards from
the fertile crescent
Harvest
Insensitive to photoperiod so flowers late
and grows throughout the summer.
Avoids the winter frost
South = winter barley (Ppd-H1)
Sowing
Harvest
Sensitive to photoperiod so flowers in the
spring when days get longer.
Avoids the heat of summer.
Cockram, J., Jones, H., Leigh, F.J., O'Sullivan, D., Powell, W., Laurie, D.A. and Greenland, A.J. (2007). Control of flowering time in temperate
cereals: genes, domestication, and sustainable productivity. J Exp. Bot. 58: 1231-1244 by permission of Oxford University Press.
© 2016 American Society of Plant Biologists
Summary and Future Perspectives
© 2016 American Society of Plant Biologists
Summary of current understanding
of circadian rhythms in plants
• Circadian rhythms are molecular time keeping
mechanisms that synchronize multiple processes with 24
hour light-dark cycles
• The circadian oscillator is a complex feedback loop
primarily based on rhythms of gene expression
• Investigating circadian rhythms requires novel
experimental approaches to capture temporal dynamics
• The circadian clock controls metabolism and key
developmental transitions
• The circadian clock is broadly similar in crop plants,
and represents a target for agronomic optimization
© 2016 American Society of Plant Biologists
There are many big questions left in
plant circadian biology
Is the oscillator
How does plant circadian
specialized in different
regulation contribute to
cell types, and do these
ecosystem dynamics?
oscillators communicate
with each other?
Can we use our
What are the
knowledge of
molecular bases circadian biology
to increase crop
How did circadian of circadian
gating?
production?
oscillators evolve?
© 2016 American Society of Plant Biologists